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Investigating Colorimetric Protein Array Assay Schemes for Detection of Recurrence of Bladder Cancer Selma Gogalic, Ursula Sauer

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, Sara Doppler and Claudia Preininger *

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Center for Health & Bioresources, AIT Austrian Institute of Technology, Konrad Lorenz Straße 24, Tulln 3430, Austria; [email protected] (S.G.); [email protected] (U.S.); [email protected] (S.D.) * Correspondence: [email protected]; Tel.: +43-5050-3527 Received: 22 November 2017; Accepted: 13 January 2018; Published: 24 January 2018

Abstract: A colorimetric microarray for the multiplexed detection of recurrence of bladder cancer including protein markers interleukin-8 (IL8), decorin (DCN), and vascular endothelial growth factor (VEGF) was established to enable easy and cheap read-out by a simple office scanner paving the way for quick therapy monitoring at doctors’ offices. The chip is based on the principle of a sandwich immunoassay and was optimized prior to multiplexing using IL8 as a model marker. Six different colorimetric assay formats were evaluated using a detection antibody (dAB) labeled with (I) gold (Au) nanoparticles (NPs), (II) carbon NPs, (III) oxidized carbon NPs, and a biotinylated dAB in combination with (IV) neutravidin–carbon, (V) streptavidin (strp)–gold, and (VI) strp–horseradish peroxidase (HRP). Assay Format (III) worked best for NP-based detection and showed a low background while the enzymatic approach, using 3,30 ,5,50 -tetramethylbenzidine (TMB) substrate, led to the most intense signals with good reproducibility. Both assay formats showed consistent spot morphology as well as detection limits lower than 15 ng/L IL8 and were thus applied for the multiplexed detection of IL8, DCN, and VEGF in synthetic urine. Colorimetric detection in urine (1:3) yields reaction signals and measurement ranges well comparable with detection in the assay buffer, as well as excellent data reproducibility as indicated by the coefficient of variation (CV 5–9%). Keywords: bladder cancer; IL8; VEGF; DCN; colorimetric protein array; carbon nanoparticles

1. Introduction Bladder cancer (BCa) is a serious malignancy of the urinary tract and prominent for its high rate of recurrence concerning 50% of all treated patients. To intervene recurrence of BCa, routine cytology and cystoscopy are done, representing the gold standard. Nevertheless, these methods are expensive, time-consuming, and invasive and lead to urinary infections in up to 16% of patients. Urine cytology has a high specificity (78–100%), although it lacks robust sensitivity (12.2–84.6%), especially for low and intermediate grade tumors, and is operator-dependent [1]. A number of urinary biomarkers (including fluorescence in-situ hybridization (FISH), ImmunoCyt, and nuclear matrix protein 22 (NMP22)) and investigational urine markers [2] also exist, but none of these markers has yet been shown to decrease the need for cystoscopy. Furthermore, biomarkers that aid the clinician in making treatment decisions are still an unmet need. Biomarkers identifying patients most likely to respond to chemotherapy for example would have enormous utility, not only reducing morbidity and improving quality of life but also significant cost savings to health providers. In previous work [3], we compiled a list of relevant bladder cancer protein markers based on extensive literature search (>1000 research articles) and prior defined inclusion and exclusion criteria. Microarray technology, a miniaturized high throughput analysis method, that can measure a high number of biomarkers in parallel was applied as a screening tool to narrow down the biomarker candidates and to evaluate the marker profiles in a clinical Biosensors 2018, 8, 10; doi:10.3390/bios8010010

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setting [4]. Ten potential protein biomarkers involved in the pathogenesis of the disease were identified and a respective biochip based on sandwich immunoassays and fluorescence detection was developed. Three (DCN, VEGF, and IL8) out of the 10 markers were significantly different (p-value < 0.05) in expression of protein levels concerning recurrent and non-recurrent BCa and were therefore considered highly suitable for detection of bladder cancer recurrence and therapy monitoring. As none of the three markers alone was specific and sensitive enough to allow for reliable detection, the three markers were combined in a multiplexed chip format, while the respective assays were transferred from a previously fluorescence-based to a colorimetric platform. Using colorimetric detection can drastically reduce instrumentation costs and enable fast and easy read-outs by a commercial office scanner, digital camera, or even smart phone. This in turn promotes broader availability and deployment at doctors’ offices and more convenient therapy monitoring, since measurement is not invasive. It is also very user-friendly, as therapy monitoring, similar to glucose monitoring, can even be performed by the patient using a smartphone. Moreover, such colorimetric multiplex assays can be efficiently implemented in point-of-care (POC) settings. In colorimetric assays, the analyte is determined with the aid of a color molecule, which is measured either as a colored solution or a colored precipitate depending on the technology platform. Using ELISA plates or microfluidic chips, the color is measured in solution in wells and lanes catalyzed by enzymes (e.g., horseradish peroxidase (HRP)) and optionally enhanced by gold nanoparticles through mechanisms, such as aggregation, controlled growth kinetics, metallization, and etching of metal nanoparticles (plasmonic ELISA) [5]. The color development relies on the biocatalytic reaction mediated modulation of local surface plasmon resonance (LSPR) properties. pELISA has enabled naked eye detection at very low concentrations of various clinically relevant biomarkers and pathogens [5]. Colored precipitates by contrast are typically detected on paper-based sensor arrays, with lateral flow devices or microarrays using enzymes, gold, or carbon nanoparticles (NPs). Enzymatic mimics, like Pd nanoparticles for detection of sarcosine in human urine samples, have also been introduced [6]. The prevalence of utilizing enzymatic labels, such as HRP and alkaline phosphatase (AP), is due to their high catalytic activity and selectivity based on specific enzyme–substrate binding. Furthermore, both the enzyme and the detection substrate are economical. Limitations of enzymatic colorimetric systems compared with detection using Au or carbon NPs are non-specific color precipitation and poor signal-to-noise ratios. Typical enzyme substrates are 3,30 ,5,50 -tetramethylbenzidine (TMB) [7] or 3,30 -diaminobenzidine tetrahydrochloride (DAB) and 5-bromo-4-chloro-30 -indolyphosphate p-toluidine salt (BCIP) [8] for HRP and AP, respectively. Generally, the detection antibody is directly labeled with the enzyme [7] or via a biotin-streptavidin bridge. Alternatively, gold and carbon NPs are used. Gold NPs are attractive labels due to their unique optical, electronic, chemical, physical, and catalytic properties [9,10], but costly in comparison with enzymes and carbon NPs. They can be produced in a wide size range between 2 and 200 nm and are easily functionalized with detection molecules forming highly stable conjugates. In a study by Jiang L. et al. [11], a colorimetric protein microarray for the serodiagnosis of TORCH infections (Toxoplasmosis, Rubella virus, Cytomegalovirus, and Herpes Simplex virus) was developed using colloidal gold particles in the size of 10.51 ± 0.24 nm coupled with the detection antibody via adsorption. The detection limit of IgM antibody was estimated to be 12.5 pg for the immunogold assay and 0.25 pg in case of the Cy3-labeled biotin-streptavidin approach [11]. Besides gold, carbon NPs of various sizes (20–200 nm) and shapes (colloidal, nano strings, nano rods, nanotubes) are used as detection labels [12,13]. Carbon NPs were firstly used in lateral flow immunoassays in the 1990s, giving proof of its suitability as a diagnostic tool in combination with image analysis [14]. Due to the high level of agreement between lateral flow devices and microarrays, carbon NPs were also introduced as detection labels in protein arrays, pioneered by Aart van Amerongen et al. [15]. For protein binding, carbon NPs were modified with neutravidin. Although the use of Au, carbon, and enzymatic labels in colorimetric strip and on-chip assays has been reported in several publications, there is no systematic work to our knowledge that compares the

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performance of different detection labels using the same type of assay and the same analyte under the same conditions. We therefore aim to evaluate HRP as well as carbon and Au nanoparticles as detection labels in an on-chip sandwich immunoassay for IL8, DCN, and VEGF to significantly improve assay performance and reach sensitivities similar to respective fluorescence assays in spiked urine. 2. Materials and Methods 2.1. Materials Chip platform used was the proprietary ARChip Epoxy (EP 02799374; US 10/490543) and ArrayIt® SuperNitro Microarray. The capture antibodies (cAB) were spotted using the ArrayIt Nanoprint contact spotter with the SMP5 pin. As assay buffer LowCross-Buffer (100 050, lot#100A895p) from Candor (Wangen, Germany) was used. Streptavidin (strp)–gold from Streptomyces avidinii (40 nm, 07211-1 mL, lot#BCBP7056V), Tween 20, sodium deoxycholate, BSA (A7906-100G, lot#SLBM3734V), 16-mercaptohexadecanoic acid (448303-5G, lot#MKBJ8615V), N-(3-Dimethylaminopropyl)-N 0 -ethylcarbodiimide hydrochloride (EDC, 03449-5G, lot#BCBN0142V) and NaCl (S5886-500G, lot#SLBM5570V), 3,30 ,5,50 -Tetramethylbenzidine (TMB2, T0565-100 mL, lot#SLBK8103V), sodium phosphate (dibasic, 30412) and silver enhancer kit (SE100-1KT) were derived from Sigma (St. Lous, MO, USA). Borate buffer (11455-90) was derived from EMS. Tris (M151-500G, lot#2243C149) was purchased from Amresco (Solon, OH, USA). EDTA (1.12029) and nitric acid (65%) were derived from Merck (Vienna, Austria). Streptavidin HRP (T20936, lot#1351296) was derived from life technologies (Carlsbad, CA, USA). S-002 (TMB1) and S-011 (O-dianisidine-based, ODI) were from Seramun (Heidesee, Germany). Human recombinant IL8 (574204) and anti-IL8 (H8A5, 511502, cAB, mouse), biotinylated anti-IL8 (mouse, E8N1, 511404) and unconjugated anti-IL8 (mouse, E8N1, 511402) were from Biolegend (San Diego, CA, USA). Neutravidin (#31000) was derived from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant human VEGF 165 protein (293-VE), human VEGF antibody (MAB293), human VEGF165 biotinylated antibody (BAF293), recombinant human decorin protein (143-DE), human decorin antibody (MAB1432) and human decorin biotinylated antibody (BAM1431) were purchased from R&D Systems (Minneapolis, MN, USA). 2.2. Chip Fabrication IL8, DCN, VEGF capture antibodies (cAB) were diluted in printing buffer (1 × PBS (pH 7.2)/0.01% sodium deoxycholate) to 0.4 g/L. The antibodies were arrayed on Supernitro slides (ArrayIt® , Sunnyvale, CA, USA) using the Arrayit Nanoprint contact spotter. The spot-to-spot distance was 500 µm. Each probe was spotted in 9 replicates in 12 identical arrays per slide at a relative humidity of 50%. To allow full immobilization of the probes the slides were kept at 4 ◦ C for at least three days. 2.3. Preparation of Immunogold Probes 1 mL of 40 nm gold particles (40 nm, CG-40-100, lot#2457227_40) from Cytodiagnostics (Burlington, VT, Canada) was centrifuged at 4 ◦ C for 15 min using 2500 g and the supernatant was discarded. 100 µL of 0.1 mM 16-MHA in water was added and incubated for 18 h at 22 ◦ C. For EDC activation, the carboxylated gold nanoparticles were centrifuged for 15 min and resuspended in 98 µL water and vortexed. 2 µL 50 mM EDC in water were added and vortexed. The particles were incubated for 30 min at 22 ◦ C. Then they were centrifuged for 15 min and resuspended in 100 µL 0.1 g/L unconjugated anti-IL8 in water. The incubation was carried out at 22 ◦ C for 2 h. After a final centrifugation step the pellet was resuspended in 100 µL TBS (20 mM Tris, 150 mM NaCl, pH 8.0) + 1% BSA. 2.4. Oxidation of Carbon Nanoparticles 200 mg of carbon Special Black 100 powder (51 nm) from Orion (Frankfurt, Germany) was dispersed in distilled water for 30 min under sonication. The resulting suspension was then refluxed in 10 mL of nitric acid at 120 ◦ C for 3 h to obtain oxidized carbon nanoparticles (oCNPs) [16]. After centrifugation

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the solid sample was washed two times with deionized water and then dried at 100 ◦ C for 18 h. Oxidation was proved by elemental analysis (SEM TM3030 with EDX, Hitachi, Feldkirchen, Germany) and Fourier-transform infrared spectra (FTIR) based on carbon black doped potassium bromide (KBr) pellets using a Spectrum 100 FT-IR instrument from PerkinElmer (Waltham, MA, USA) [17]. 2.5. Antibody Coupling to oCNPs 1% (w/v) suspension of oCNPs was prepared and sonicated for 30 s. Then 100 µL of 0.5 g/L unconjugated anti-IL8, anti DCN or anti VEGF was added dropwise to 150 µL of 5-fold dilution of oCNPs in 5 mM sodium borate coupling buffer (pH 8.8) (0.2% w/v) and allowed to react for 18 h at 4 ◦ C under gentle stirring. 2.6. Modification of Carbon NPs with Neutravidin 1% (w/v) suspension of carbon NPs (used as received) was prepared and sonicated for 5 min. Then the 1% suspension was diluted 5-fold in 5 mM borate buffer and again ultrasonicated for 5 min. Next, 0.35 mg neutravidin was added to 1 mL of diluted carbon NP suspension and incubated for 3 h at RT on an over-head shaker (Biorotator, Biospec Products) [18]. Both probes from Sections 2.5 and 2.6 were then washed (15 min, 13,600 g) at least three times with washing buffer (5 mM borate buffer + 1% BSA + 0.002% NaN3 ). Finally, the supernatant was removed and the pellet resuspended in storage buffer (100 mM borate buffer + 1% BSA + 0.002% NaN3 ) at a final concentration of 0.2% and stored at 4 ◦ C till usage. Before use in the on-chip assay, the carbon particles were sonicated for 10 s. 2.7. Chip Processing Surface blocking was done for 45 min in 1× PBS (pH 7.2)/0.1% Tween 20 in order to remove any unbound probes and to deactivate reactive surface groups. Slides were washed twice in 1× PBS (pH 7.2) and dried using compressed air. Slides were mounted into a Hybridization Cassette 4 × 16 (ArrayIt Corporation, Sunnyvale, CA, USA). 12 arrays/wells (7 mm × 7 mm) per slide were used. Each array was incubated with 50 µL calibration standard for 2.5 h. Spiked samples were prepared by serial dilution of mixes of analytes in assay buffer or synthetic urine (certified drug free urine (88121-CDF(F)) UTAK Laboratories Inc., Santa Clara, CA, USA) (1:3) (1 part urine, 2 parts assay buffer). Then the slides were washed three times with 1× PBS (pH 7.2)/0.1% Tween 20. 2.7.1. Detection Antibody Labeled with Carbon-and Gold NPs Slides were incubated for 45 min with 50 µL oCNPs labeled dAB (1:5 diluted in assay buffer) and gold NPs labeled dAB (1:5 diluted in buffer: 5% BSA, 0.15 M NaCl, 0.01 M sodium phosphate (pH 7) and 0.05% Tween). Finally, the slides (treated with oCNPs) were washed twice with 1× PBS (pH 7.2)/0.1% Tween 20, twice in 1× PBS (pH 7.2) and once in distilled water. Slides incubated with gold-labeled dAB were in addition incubated with 0.05 M EDTA for 5 min before silver staining (10 min, Section 2.8). All incubation steps were carried out on the orbital shaker (Stovall, Greensboro, NC, USA) at maximum speed and at room temperature. After washing the slides were dried using compressed air and stored in the dark until scanning. 2.7.2. Detection via Streptavidin/Neutravidin-Biotin Binding Slides were treated with 50 µL biotinylated dAB and further incubated with either neutravidin–carbon (1:20 dilution in assay buffer), strp-HRP (1:100 in assay buffer) or strp–gold (1:5 in buffer: 5% BSA, 0.15 M NaCl, 0.01 M sodium phosphate dibasic (pH 7.0) and 0.05% Tween 20) for 45 min. Slides incubated with strp–gold were washed with 1× PBS (pH 7.2)/0.1% Tween 20, 1× PBS (pH 7.2), distilled water and incubated with 0.05 M EDTA for 5 min before silver staining (10 min, Section 2.8). The slides incubated with strp-HRP were also washed as before and treated with substrate

Slides were treated with 50incubated µLwith biotinylated dAB and further incubated with either neutravidin– Slides were treated with 50dAB µL biotinylated dAB andincubated further incubated with either neutravidin– Slides were with 50 µL biotinylated dAB and further incubated with either neutravidin– carbon (1:20 dilution in assay buffer), strp Slides were treated with 50 and µL biotinylated dAB and further with either neutravidin– lides treated with 50 µL biotinylated and further incubated with either neutravidin– with 50were µL biotinylated dAB and further incubated either neutravidin– eated with 50 treated µL biotinylated dAB further with either neutravidin– carbon (1:20 dilution in assay buffer), strp-HRP (1:100 in assay buffer) or strp–gold (1:5 inNaCl, buffer:0.01 M sodium phos carbon (1:20 dilution in assay buffer), strp-HRP (1:100 in assay buffer) or strp–gold (1:5 in buffer: rs 2018, 8, x 5 of 13 bon dilution instrp-HRP assay buffer), strp-HRP (1:100 inorassay buffer) or in strp–gold in(1:5 buffer: 5% 0.15 M carbon (1:20 dilution in assay buffer), strp-HRP (1:100 in(1:5 assay buffer) or strp–gold inBSA, buffer: n (1:20 dilution in assay buffer), strp-HRP (1:100 assay buffer) strp–gold (1:5(1:5 in buffer: assay buffer), strp-HRP (1:100 in assay or in strp–gold inor(1:5 buffer: ion in(1:20 assay buffer), (1:100 inbuffer) assay buffer) strp–gold buffer: 5% BSA, 0.15 M NaCl, 0.01 M sodium phosphate dibasic (pH 7.0) and 0.05% Tween 20) for 45 min. with strp–gold were w 5% BSA, 0.15 M NaCl, 0.01 M sodium phosphate dibasic (pH 7.0) and 0.05% Tween 20) for 45 min. BSA, 0.15 M NaCl, 0.01 M0.01 sodium phosphate dibasic (pH 7.0) and 0.05% Tween 20) 45 for min. Slides incubated 5% BSA, 0.15 M NaCl, M sodium phosphate dibasic (pH 7.0) and 0.05% Tween 45 min. MMNaCl, 0.01 M dibasic sodium phosphate dibasic 7.0)Tween and 0.05% Tween 20) for for 4520) min. 1A, M0.15 sodium phosphate (pH 7.0)(pH and 0.05% Tween 20) for 45 min. aCl, 0.01 sodium phosphate dibasic 7.0) and(pH 0.05% 20) for 45 min. Detection via Streptavidin/Neutravidin-Biotin Binding Slides incubated with strp–gold were washed with 1× PBS (pH Tween 20,PBS 1× PBS (pH incubated with 0.05 Slides incubated with strp–gold were washed with 1× 1× PBS (pH 7.2)/0.1% Tween 20,(pH 1× (pHand Slides incubated with strp–gold were washed with 1× PBS (pH Tween 20, 1× 20, PBS (pH 7.2), distilled water Slides incubated with strp–gold were washed with 1× PBS (pH 7.2)/0.1% Tween 1× PBS lides incubated with strp–gold were washed with 1×Tween PBS (pH 7.2)/0.1% Tween 20, 1×7.2)/0.1% PBS (pH h strp–gold were washed with 1× PBS (pH 7.2)/0.1% 20, 1×7.2)/0.1% PBS (pH ted with strp–gold were washed with 1× PBS (pH 7.2)/0.1% Tween 20, PBS (pH ides were treated with 50 µL biotinylated dAB and further incubated with either neutravidin– 7.2), distilled water and incubated with 0.05 M EDTA for 5 min before silver staining (10 min, Section 7.2), distilled water and incubated with 0.05 M EDTA for 5 min before silver staining (10 min, Section Biosensors 2018, 8, 10 5 of 13with strp-HRP we ), distilled water and 0.05 M0.05 EDTA for 5 min before silver staining (10 min, 2.8).Section The slides incubated 7.2), distilled water andMincubated with M for 5 min before silver staining (10Section min, water and incubated with 0.05 M EDTA forEDTA 5silver min before silver staining (10 min, Section ncubated with 0.05 M incubated EDTA for 5with min before silver staining (10 min, Section ristilled and incubated with 0.05 EDTA for 5 min before staining (10 min, Section (1:20 dilution in assay buffer), strp-HRP (1:100 in assay buffer) or strp–gold (1:5 in buffer: 2.8). The slides incubated strp-HRP were also washed astreated before and treated with substrate (TMB 2.8). The slides incubated with strp-HRP were also washed as before and treated with substrate ). The slides incubated with strp-HRP were also washed as before and treated with substrate (TMB or ODI) for 15(TMB min. At the end all slides w 2.8). The slides incubated with strp-HRP were also washed as before and with substrate (TMB he slides incubated with strp-HRP were also washed as before and treated with substrate (TMB with strp-HRP were also washed as before and treated with substrate (TMB ubated with strp-HRP were also washed aswith before and treated with substrate (TMB A, 0.15 M NaCl, 0.01 M sodium phosphate dibasic (pH 7.0) and 0.05% Tween 20)twice for 45 with min. or ODI) for 15 min. At the end all slides were washed 1× PBS (pH 7.2)/0.1% Tween 20, and in distilled w or ODI) for 15 min. At the end all slides were washed twice with 1× PBS (pH 7.2)/0.1% Tween 20, ODI) for 15 min. At the end all slides were washed twice with 1× PBS (pH 7.2)/0.1% Tween 20, twice in 1× PBS (pH 7.2) or ODI) for 15 min. At the end all slides were washed twice with 1× PBS (pH 7.2)/0.1% Tween 20, DI) for 15 min. At the end all slides were washed twice with 1× PBS (pH 7.2)/0.1% Tween 20, he endthe all end slides twice with PBS 1× (pH 7.2)/0.1% Tween Tween 20, n. At all were slideswashed were washed twice1×with PBS (pH 7.2)/0.1% 20, (TMB or ODI) for 15 min. At the end all slides were washed twice with 1× PBS (pH 7.2)/0.1% Tween ides incubated with strp–gold were washed with 1× PBS (pH 7.2)/0.1% Tween 20, 1×steps PBS (pH twice in 1× PBS (pH 7.2) and in distilled water. All incubation steps were carried out on the orbital twice in 1× PBS (pH 7.2) and in distilled water. All incubation were carried out on the orbital ice in 1×and PBS 7.2) and inand distilled water. All incubation steps were carried out the orbital shaker (Stovall) at maximum speed at room twice in(pH 1×(pH PBS (pH 7.2) distilled water. All incubation steps were carried out on the orbital in 7.2) 1× 7.2) and in distilled water. All incubation steps carried out on on the orbital and inPBS distilled water. All incubation steps were carried out onwere the orbital H in distilled water. Allin incubation steps were carried out on the orbital stilled water and incubated with M EDTA for 5and minin before silverwater. staining min, Section 20, twice in 10.05 × PBS (pH 7.2) distilled All(10 incubation steps were carried out on the orbital shaker (Stovall) at maximum speed at room temperature. After washing theusing slides were dried using shaker (Stovall) at at maximum speed at room temperature. After washing the slides were dried using aker (Stovall) at maximum speed at room temperature. After washing the slides were dried compressed air and stored in the dark until shaker (Stovall) at maximum speed at room temperature. After washing the slides were dried using r (Stovall) at maximum speed room temperature. After washing the slides were dried using mum speed at room temperature. After washing the slides were dried using maximum speed at room temperature. After washing the slides were dried using he slides incubated with strp-HRP were washed as before treated with substrate (TMB shaker (Stovall) atalso maximum speed atand room temperature. After washing the slides were dried using compressed air and stored in the dark until scanning. compressed air and stored in the dark until scanning. mpressed air and stored in the dark until scanning. compressed airscanning. and stored in until the dark until scanning. ressed stored inall the dark scanning. d the dark until d in stored inand the dark until scanning. I) for 15air min. At the end slides PBS scanning. (pH 7.2)/0.1% Tween 20, compressed airwere andwashed stored twice in thewith dark1×until 2.8. Silver Staining n 1× PBS (pH 7.2) and in distilled water. All incubation steps were carried out on the orbital 2.8. Silver Staining 2.8. Silver Staining .lver Silver Staining 2.8. Silver Staining Staining (Stovall) at maximum speed at room temperature. After washing the slides were dried using 2.8. Silver Staining For the silver enhancement the excess essed air and stored in the dark until scanning. For the silver enhancement the excess solution was wiped off, 50 µL/well silver For the silver enhancement the excess solution was wiped off, 50 µL/well silver solution werewere For silver enhancement the excess solution was wiped off, 50off, µL/well silver solution were silver added andsolution incubated for 10 min. Finally, th For the silver enhancement the excess solution was wiped 50 silver µL/well silver solution were or thethe silver enhancement the excess solution was wiped off, 50 µL/well solution were cement the excess solution was wiped off, 50 µL/well silver solution were enhancement the excess was wiped off, 50 µL/well silver solution were Forsolution the silver enhancement the excess solution was wiped off, 50 µL/well solution were added and incubated for 10 min. Finally, thewith slides were fixed with sodium thiosulfate solution added and incubated for 10 min. Finally, the slides were fixed with 2.5%2.5% sodium thiosulfate solution and incubated for 10 min. Finally, the slides were fixed 2.5% sodium thiosulfate solution for 3 min and washed with dH2O. The slide added and incubated for 10 min. Finally, the slides were fixed with 2.5% sodium thiosulfate solution dded and incubated for 10 min. Finally, the slides were fixed with 2.5% sodium thiosulfate solution 10 min. Finally, the slides were fixed with 2.5% sodium thiosulfate solution ted for 10 min. Finally, the slides were fixed with 2.5% sodium thiosulfate solution added and incubated for 10 min. Finally, the slides were fixed with 2.5% sodium thiosulfate solution ver Staining for 3 min and washed with dH 2 O. The slides were dried using compressed air. for 3 min and washed with dH 2 O. The slides were dried using compressed air. 3 min and washed with dH 2 O. The slides were dried using compressed air. for 3 washed min washed with dH 2O. compressed The slides were dried compressed air. min and with 2dried O. The slides were dried using compressed air. using th dH 2O. The slides using air. hed with dH2and O. The slides were dried using compressed air.using forwere 3dH min and washed The slides were dried compressed air. 2 O. off, or the silver enhancement the excess solutionwith wasdH wiped 50 µL/well silver solution were 2.9. Data Analysis incubated forData 10 min. Finally, the slides were fixed with 2.5% sodium thiosulfate solution 2.9. Analysis Analysis .and Data 2.9.Analysis Data2.9. Analysis ata Analysis 2.9.Data Data Analysis The colorimetric signal intensity was m min and washed with dH2O. The slides were dried using compressed air. The colorimetric signal intensity was measured using an office scanner (Epson Perfection The colorimetric signal intensity was measured using an office scanner (Epson Perfection V330V330 The colorimetric signal intensity was measured using an office (Epson Perfection V330 Photo). Data were analyzed The colorimetric signal intensity was measured using an scanner office scanner (Epson Perfection V330 The colorimetric intensity was measured using anwas office scanner (Epson Perfection V330 nal intensity wassignal measured using an office scanner (Epson Perfection V330 tric signal intensity was measured using an office scanner (Epson Perfection V330 The colorimetric signal intensity measured using an office scanner (Epson Perfection V330 with the Gene ta Analysis Photo). Data were analyzed with the Genepix 6.0 software. The mean signal values were calculated Photo). Data were analyzed with the Genepix 6.0 software. The mean signal values were calculated oto). Data were analyzed with the Genepix 6.0 software. The mean signal values were calculated from nine background corrected data poin Photo). Data were analyzed with the Genepix 6.0 software. The mean signal values were calculated ). Data were analyzed with the Genepix 6.0 software. The mean signal values were calculated ed with the Genepix 6.0 software. The mean signal values were calculated analyzed with the Genepix 6.0 software. The mean signal values were calculated Photo). Data were analyzed with the Genepix 6.0 software. The mean signal values were calculated from nine background corrected data points. Data points that were out of the mean signal values ± from nine background corrected data points. Data points that were out of the mean signal values ± m nine background corrected data points. Data points that were out of the mean signal values ± the standard deviation (SD) were excluded from ninepoints. background corrected data points. points that were out of the signal ± nine background corrected data points. Data points that were out of the mean signal values ± values orrected data Data points that were out of the mean signal values ± that ound corrected data points. Data points that were out of the mean signal values ± mean he colorimetric signal intensity was measured using anData office scanner (Epson Perfection V330 from nine background corrected data points. Data points were out of the mean signal values ± the standard deviation (SD) were excluded in an outlier test. Calibration curves were set up with the standard deviation (SD) were excluded in an outlier test. Calibration curves were set up with . standard Data were analyzed with the Genepix software. The mean signal values were calculated deviation (SD) were excluded in an outlier test. Calibration were with OriginPro 8 using thewith logistic fit. The limit o the standard deviation (SD) were excluded incurves an outlier test. curves were set up with andard deviation (SD) were excluded in an outlier test. Calibration curves were set set up up with SD) were excluded in an outlier test.6.0Calibration were set upoutlier with ation (SD) were excluded in andeviation outlier test. Calibration curves were setcurves up with the standard (SD) were excluded in anCalibration test. Calibration curves were set up OriginPro 8 using the logistic fit. The limit of detection (LOD) and dynamic range of the comparative OriginPro 8 using the logistic fit. The limit of detection (LOD) and dynamic range of the comparative ine OriginPro background corrected data points. Data points that were out of the mean signal values ± iginPro 8 using the logistic fit. The limit of detection (LOD) and dynamic range of the comparative IL8 assays were determined 8The using the logistic fit. The limit of detection (LOD) and dynamic range of the comparative nPro 8 using the logistic fit. The limit of detection (LOD) and dynamic range of the comparative istic fit. The limit of detection (LOD) and dynamic range of the comparative the logistic fit. limit of detection (LOD) and dynamic range of the comparative OriginPro 8 using the logistic fit. The limit of detection (LOD) and dynamic range of the comparative visually. ndard deviation (SD) were excluded in an outlier test. Calibration curves were set up with IL8 assays were determined visually. IL8 assays were determined visually. 8etermined assays were determined visually. IL8 assays were determined visually. says were determined visually. ed visually. visually. IL8 assays were determined visually. Pro 8 using the logistic fit. The limit of detection (LOD) and dynamic range of the comparative 3. Results and Discussion ays 3. were determined visually. 3.3.Results Discussion 3. Results and and Discussion Results and Discussion Results and Discussion Results and Discussion and Discussion nults scussion 3.1. Colorimetric Assay Schemes ults and Discussion3.1. Colorimetric Assay Schemes 3.1. Colorimetric Assay Schemes 3.1. Colorimetric Assay Schemes .ssay Colorimetric Assay Schemes 3.1. Colorimetric Assay Schemes olorimetric Assay Schemes emes Schemes Six colorimetric immunoassay scheme lorimetric Assay Schemes Sixcolorimetric colorimetric immunoassay schemes as described in Figure were terms Six immunoassay schemes as described in Figure 1a1a were investigated ininterms Six colorimetric immunoassay schemes described in 1a investigated in of ofof signal intensity colorimetric immunoassay schemes as inasFigure 1a were investigated in terms of sensitivity, reproducibility, Six colorimetric immunoassay schemes as1adescribed in Figure 1aFigure were investigated ininvestigated terms of terms ixSix colorimetric immunoassay schemes as in Figure 1a were investigated in terms of munoassay schemes as described in Figure 1adescribed were investigated in terms of ric immunoassay schemes as described indescribed Figure were investigated in terms of were sensitivity, reproducibility, signal intensity, and assay time to finally select the most sensitive onemultiplexed biom sensitivity, reproducibility, signal intensity, and assay time to finally select the most sensitive one sensitivity, reproducibility, signal intensity, and assay time to finally select the most sensitive one for nsitivity, reproducibility, signal intensity, and assay time to finally select most thefordevelopment of for the sensitivity, reproducibility, signal intensity, assay time to finally select the most one for ivity, reproducibility, intensity, andto assay time to1a finally select thethe most sensitive oneone for y, signal intensity, andsignal assay time to finally select the most sensitive one for signal intensity, and assay finally select the most sensitive one forsensitive xucibility, colorimetric immunoassay schemes astime described inand Figure were investigated in terms of sensitive for the development of the multiplexed biomarker chip. Nitrocellulose slides were used as the chip the development of the multiplexed biomarker chip. Nitrocellulose slides were used as the chip the development of the multiplexed biomarker chip. Nitrocellulose slides were used asFor thecost chipreasons, we chose IL8 fo vity, reproducibility, signal intensity, and assaybiomarker time to finally select the sensitive oneused for of the multiplexed biomarker chip. Nitrocellulose slides were the chip platform. the development of the multiplexed chip. Nitrocellulose slides were used as the chip evelopment of the multiplexed biomarker chip. Nitrocellulose slides were used as as the chip multiplexed biomarker chip. Nitrocellulose slides were used asmost the chip ofdevelopment the multiplexed biomarker chip. Nitrocellulose slides were used as the chip platform. For cost reasons, we chose IL8 for evaluation of the proposed assay schemes. IL8 capture platform. For cost reasons, we chose IL8 for evaluation of the proposed assay schemes. IL8 capture velopment of the multiplexed biomarker chip. Nitrocellulose slides were used as the chip platform. For cost reasons, we chose IL8 for evaluation of the proposed assay schemes. IL8 capture tform. For cost reasons, we chose IL8 for evaluation of the proposed assay schemes. IL8 capture platform. For cost reasons, we chose IL8 for evaluation of the proposed assay schemes. IL8 capture rm. For cost reasons, we chose IL8 for evaluation of the proposed assay schemes. IL8 capture s, we chose IL8 for evaluation of the proposed assay schemes. IL8 capture reasons, we chose IL8 for evaluation of the proposed assay schemes. IL8 capture antibodies (cAB ) were spotted onto th

m. For cost reasons, we chose IL8 for evaluation of the proposed assay schemes. IL8 capture

antibodies (cAB )the were spotted onto the chip bind ) solution present insolution the solution antibodies (cAB )bind were spotted the chip to bind IL8 )sample present in the sample solution antibodies (cAB )( were spotted onto the bind IL8 (in(the ) present insolution the sample solution (Stepof2).the binding, the ch tibodies (cAB )chip were onto chip toonto bind IL8 (chip )solution present (Step 2).sample For detection antibodies (cAB )spotted were spotted onto the chip to bind IL8 ( to )(IL8 present in the sample odies (cAB )the were spotted onto the chip bind (the )to present in the sample ere spotted onto to bind IL8 present in sample ) were spotted onto the chip to IL8 ( )to )IL8 present in the sample solution dies (cAB ) were spotted onto the chip to bind IL8 ( ) present in the sample solution (Step 2). For detection of the binding, the in a step, 3rd incubated with a detection antibody (Step detection of binding, chip was, aantibody 3rd incubated with a detection antibody For detection of the binding, the chip inincubated ain 3rd step, incubated a detection antibody that waswith NPs (I–III) ep 2). detection ofFor the binding, the chip was, a 3rd step, with awith detection antibody (Step 2). For detection of binding, the chip was, in achip 3rd step, incubated awith detection antibody 2). detection of2). the binding, the chip was, inincubated ainthe 3rd step, incubated with astep, detection antibody he For binding, the chip was, inwas, athe 3rd incubated with awas, detection tion ofFor the binding, the chip instep, a the 3rd step, with awas, detection antibody that was either labeled

). For detection of the binding, the chip was, in a 3rd step, incubated with a detection antibody

that was either labeled with NPs (I–III) or incubation biotinylated (IV–VI ). While incubation an (I) gold (AuNPs that was either labeled with NPs (I–III) or biotinylated (IV–VI ).incubation While incubation withwith an at was either labeled with NPs (I–III) or biotinylated (IV–VI ). While incubation with an antibody with that was either labeled with NPs (IV–VI ).incubation While with anlabeled was either labeled with NPs (I–III) or (I–III) biotinylated ). with While with an either labeled with NPs (I–III) orbiotinylated biotinylated (IV–VI ).with While with an antibody labeled with NPs (I–III) or biotinylated (IV–VI ).or While incubation an abeled with NPs (I–III) or biotinylated (IV–VI ).(IV–VI While anincubation as either labeled with NPs (I–III) or biotinylated (IV–VI ). While incubation with an antibody labeled with (I) gold (AuNPs ), (II) carbon (CNPs ), or (III) oxidized carbon antibody labeled with (I) gold (AuNPs ), (II) carbon (CNPs ), or (III) oxidized carbon tibody labeled with gold (AuNPs ),(II) (II) carbon (CNPs ),(III) or (III) carbon antibody labeled with (I) gold (AuNPs ), (II) carbon ), oxidized oroxidized (III)carbon oxidized carbon (oCNPs ) ) allows direct de ody labeled with (I) gold (AuNPs (II) carbon or (III) carbon (I) gold (AuNPs ),(CNPs carbon (CNPs ),),or oxidized nanoparticles I)with gold (AuNPs ),(I) (II) carbon (CNPs ), or (III) carbon (I) gold (AuNPs ), (II) carbon ),(CNPs oroxidized oxidized carbon dy labeled with (I)with gold (AuNPs ), (II)),carbon (CNPs ),(III) or (CNPs (III) oxidized carbon nanoparticles (oCNPs ) allows direct detection of the binding, the use of a biotinylated antibody nanoparticles (oCNPs ) allows direct detection of the binding, the use of a biotinylated antibody noparticles (oCNPs ) allows direct detection of the binding, the use of a biotinylated antibody requires additional incubation steps for si nanoparticles (oCNPs ) allows direct detection of the binding, the use of a biotinylated antibody particles (oCNPs ) allows direct detection of the binding, the use of a biotinylated antibody allows direct detection of the binding, the use of a biotinylated antibody requires additional incubation ) allows direct detection of the binding, the use of a biotinylated antibody NPs ) allows direct detection of the binding, the use of a biotinylated antibody articles (oCNPs ) allows direct detection of the binding, the use of a biotinylated antibody requires additional incubation steps for signal detection. In Format (IV), Step 4, the biotinylated requires additional incubation steps for signal detection. In Format (IV), Step 4, the biotinylated quires additional incubation steps signal detection. In Format Step 4,Step the biotinylated requires additional incubation steps for signal detection. In Format (IV), 4, the biotinylated esincubation additional incubation steps fordetection. signal detection. In 4, Format Step 4, the biotinylated steps for signal In Format (IV), Step 4, the biotinylated detection antibody reactsreacts with with neutravidin bation steps incubation for signal detection. Infor Format (IV), Step the biotinylated al steps for steps signal detection. In Format (IV), Step 4,(IV), the biotinylated es additional for signal detection. In Format (IV), Step 4,(IV), biotinylated detection antibody detection antibody reacts with neutravidin adsorbed on carbon nanoparticles ( ); in Format (V), detection antibody reacts with neutravidin adsorbed on carbon nanoparticles ( ); in Format (V), neutravidin adsorbed on nanoparticles in (V), coupled with Auwith Au ( on antibody with neutravidin adsorbed oncarbon carbon nanoparticles ( (); nanoparticles in( Format Format (V), antibody reacts with neutravidin adsorbed nanoparticles ( ); );( Format in streptavidin Format (V), is(V), detection antibody reacts with neutravidin adsorbed onnanoparticles ); in(V), Format ion antibody reacts with neutravidin on on carbon in with neutravidin adsorbed on carbon nanoparticles ( carbon );carbon Format (V), ytection reacts withreacts neutravidin adsorbed onadsorbed carbon nanoparticles (in in););Format (V), streptavidin is coupled ); vidin iswith (Format in (VI), streptavidin is linked tolinked HRP (tolinked is);(coupled with Au (is(VI), in Format (VI), streptavidin linked to HRP ( ).treated ).(I) and (V) are treate is coupled with Au );linked in);streptavidin Format streptavidin is(tolinked eptavidin isstreptavidin coupled with Au ); in Format is to (is Additionally, streptavidin is(inwith coupled with Au (streptavidin ); (in Format (VI), streptavidin is HRP ( ).(I)HRP ).((V)Formats avidin iscoupled ((VI), );Format in Format (VI), streptavidin HRP ). to with Au (coupled );streptavidin Format streptavidin is linked to HRP (is upled Au (with );inAu in Format (VI), streptavidin is linked to(VI), ().Additionally, ). ).HRP );Au (VI), to HRP (HRP ).linked Formats and are onally, Formats (I) and (V) are treated with silver (Ag), while Format (VI) is (Ag), further Additionally, Formats (I) and (V) are treated with silver (Ag), while Format isdifferent further processed Additionally, Formats (I)while and (V) are treated with silver while Format (VI) (VI) is further processed ditionally, (I) and (V) are treated with silver (Ag), while Format (VI) is further processed with enzyme Additionally, Formats (I) and (V) are treated with silver (Ag), while Format (VI) is further processed ionally, Formats (I) and (V) are treated with silver (Ag), while Format (VI) isprocessed further processed and (V) areFormats treated with silver (Ag), Format (VI) is further processed mats (I) and (V) are treated with silver (Ag), while Format (VI) is further processed with silver (Ag), while Format (VI) is further processed with different enzyme substrates (Step 5). substrates (Step 5). ifferent enzyme substrates (Step 5). with different enzyme substrates (Step 5). with different enzyme substrates (Step 5). th different enzyme substrates (Step 5). AuNPs were modified with different enzyme substrates (Step 5). using 16-mercaptohexadecanoic acid (MHA) (I), and different enzyme substrates (Step 5). modified bstrates (Step 5).(Step yme substrates 5).AuNPs were the antibody was using 16-merca uNPs were modified using 16-mercaptohexadecanoic acid (MHA) (I), and the antibody was AuNPs were modified using 16-mercaptohexadecanoic acid (MHA) (I),bound and the antibody AuNPs were modified using 16-mercaptohexadecanoic acid (MHA) (I), antibody and the antibody was was AuNPs were modified using 16-mercaptohexadecanoic acid (MHA) (I), and the antibody was to the via EDC, a zero length AuNPs were modified using 16-mercaptohexadecanoic acid (MHA) (I), and the was AuNPs were modified using 16-mercaptohexadecanoic acid (MHA) (I), and the antibody was ed using 16-mercaptohexadecanoic acid (MHA) (I), the antibody was modified using 16-mercaptohexadecanoic acid (MHA) (I), and the antibody was bound to the particle via EDC, a and zero length crosslinker that covalently couples theparticle carboxylic to the particle via EDC, a zero length crosslinker that covalently couples the carboxylic group bound toof the particle via EDC, acrosslinker zero length crosslinker that covalently couples the carboxylic group groups of the ant bound to the particle via EDC, a zero length crosslinker that covalently couples the carboxylic group toathe the particle via EDC, aEDC, zero crosslinker that covalently couples the carboxylic group of untreated MHA to for the amino bound to particle via alength zero length that covalently couples the carboxylic group dund toto the particle via EDC, zero length crosslinker that covalently couples the carboxylic group EDC, zero length crosslinker that covalently couples the carboxylic group cle via EDC, athe zero length that covalently couples the carboxylic group group MHA to the amino groups of the antibody. CNPs were either used antibody HA amino groups ofacrosslinker the antibody. CNPs were either used untreated for antibody of MHA toofgroups the amino groups of the antibody. CNPs were either used untreated for antibody of amino MHA to the amino groups of the antibody. CNPs were either used untreated for antibody MHA to groups of the antibody. CNPs were either used untreated for antibody (II) or oxidized (oCNPs) wi of the amino of the CNPs were either used untreated forimmobilization antibody HA to MHA the amino groups the antibody. CNPs were either used untreated for antibody groups of(II)the the antibody. CNPs were either used untreated for antibody mino groups of the antibody. CNPs were either used untreated for immobilization (II) or oxidized (oCNPs) with nitric acid to introduce reactive quinone or conjugated bilization orto oxidized (oCNPs) with nitric acidantibody. to introduce reactive quinone orantibody conjugated immobilization (II) or oxidized (oCNPs) with nitric acid to introduce reactive quinone or conjugated immobilization (II) or oxidized (oCNPs) with nitric acid to introduce reactive quinone or conjugated mobilization (II) or oxidized (oCNPs) with nitric acid to introduce reactive quinone or conjugated ketone groups for antibody binding immobilization (II) or oxidized (oCNPs) with nitric acid to introduce reactive quinone or conjugated bilization (II) or oxidized (oCNPs) with nitric acid to introduce reactive quinone or conjugated dized (oCNPs) with nitric acid to introduce reactive quinone or conjugated ) groups or oxidized (oCNPs) with nitric[17] acid introduce reactive quinone or conjugated ketone groups fortoThe antibody binding (III). The neutravidin/streptavidin–biotin bridge was [17] exploited [17] for antibody binding (III). neutravidin/streptavidin–biotin bridge was ketone groups [17] for antibody binding (III). The neutravidin/streptavidin–biotin bridge was ketone groups [17] for antibody binding (III). The neutravidin/streptavidin–biotin bridge was one groups [17] for antibody binding (III). The neutravidin/streptavidin–biotin bridge was was exploited in Formats (IV) and (V), so neutra ketone groups [17] for antibody binding (III). The neutravidin/streptavidin–biotin bridge e7] [17] for antibody binding (III). The neutravidin/streptavidin–biotin bridge was antibody binding (III). The neutravidin/streptavidin–biotin bridge was edgroups in Formats (IV) and (V), so neutravidin and streptavidin, respectively, werebridge adsorbed onto for antibody binding (III). The was in Formats (IV) andneutravidin/streptavidin–biotin (V), so neutravidin and streptavidin, respectively, were adsorbed onto the NPs. exploited in(V), Formats (IV) and (V), so neutravidin and streptavidin, respectively, were adsorbed onto exploited in Formats and (V), so neutravidin and streptavidin, respectively, were adsorbed onto(I) s. ploited in Formats and so(IV) neutravidin and streptavidin, respectively, were adsorbed onto the NPs. exploited in (IV) Formats (IV) and (V), so neutravidin and streptavidin, respectively, were adsorbed onto ited in Formats and (V), so neutravidin and streptavidin, respectively, were adsorbed onto and (V), so neutravidin and streptavidin, respectively, were adsorbed onto ats (IV) and (V), so (IV) neutravidin and streptavidin, respectively, were adsorbed onto In total, we evaluated two colorimetric assay schemes using Au nanoparticles (Formats and the NPs. the NPs. NPs. Ps. the NPs. (V)), three using carbon nanoparticles (Formats (II)–(IV)), and one enzymatic approach (Format (VI)). A scheme of NPs modifications is shown in Figure 1b.

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In total, we evaluated two colorimetric assay schemes using Au nanoparticles (Formats (I) a (V)), three using carbon nanoparticles (Formats (II)–(IV)), and one enzymatic approach (Format (VI 6 of 13 A scheme of NPs modifications is shown in Figure 1b.

(a)

(b)

Figure 1. (a) Schematic presentation of six distinct assay formats for colorimetric detection: (I) cAB + IL8 + gold-labeled dAB, (II) cAB + IL8 + carbon-labeled dAB, (III) cAB + IL8 + oxidized carbon-labeled dAB, (IV) cAB + IL8 + biot. dAB + neutravidin–carbon, (V) cAB + IL8 + biot. dAB + strp–gold, and (VI) cAB + IL8 + biot. dAB + strp-HRP + enzyme substrate. Slides treated via gold nanoparticles were further stained by silver (Ag). (b) Illustration of the antibody (in orange) coupling process using gold (red) and carbon (black) nanoparticles.

3.2. Assay Schemes with Nanoparticle-Labeled Detection Antibody

A comparative study was conducted using anti-IL8 labeled with Au and carbon NPs (Figure (I)–(III)) to directly detect IL8 in the concentration range of 1 ng/L to 98.4 µg/L. Commercially available NPs were utilized to guarantee a high percentage of regularly shap NPs preventing diffuse signals and generating well-confined and consistent spots [13]. Nitrocellulo with apresentation 150 µm nitrosylated polysaccharide and a 0.20 detection: µm porosity was+applied. In order to wo Figure 1. (a) Schematic of six distinct assay formatslayer for colorimetric (I) cAB with colloidal stable [19] and sensitive AuNPs, 40-nm-sized particles were chosen. This is in go IL8 + gold-labeled dAB, (II) cAB + IL8 + carbon-labeled dAB, (III) cAB + IL8 + oxidized carbon-labeled agreement with Nath et al. [20], who investigated the effect of particle size on the analytical sensitiv dAB, (IV) cAB + IL8 + biot. dAB + neutravidin–carbon, (V) cAB + IL8 + biot. dAB + strp–gold, and of andAB optical biosensor for streptavidin–biotin as a via model (VI) cAB + IL8 + biot. + strp-HRP + enzyme substrate. Slides treated gold receptor–ligand nanoparticles werepair. The authors show further stained bythat silver (b) Illustration of the antibody (inby orange) coupling process using gold the(Ag). sensitivity of the sensor increases a factor of three as size of Au NPs increases in the test (red) and carbon range (black)of nanoparticles. 13–47 nm. AuNPs have been coupled with anti-IL8 using MHA and carbodiimide [21]. MHA se 3.2. Assay Schemes with Nanoparticle-Labeled Detection Antibody assembles on the Au particle forming a monolayer. Its carboxy group is subsequently activated w EDC to covalently bind IL8 through the amino groups present in the amino acids (arginine a A comparative study was conducted using anti-IL8 labeled with Au and carbon NPs (Figure 1, lysine). Alternatively, the antibody might only be mixed with the AuNPs and adsorb on the partic (I)–(III)) to directly detect IL8 in the concentration range of 1 ng/L to 98.4 µg/L. via its thiol functions (cysteine). This was realized by Wang et al. [22], who coupled 40 nm A Commercially available NPs were utilized to guarantee a high percentage of regularly shaped particles to an anti-aflatoxin M1 antibody via adsorption. NPs preventing diffuse signals and generating well-confined and consistent spots [13]. Nitrocellulose In our study, the adsorption of antibodies on plain Au nanoparticles was not successful and t with a 150 µm nitrosylated polysaccharide layer and a 0.20 µm porosity was applied. In order to work coupling antibody to Au via MHA led to mixed results with sometimes poor signals (Detecti with colloidal stable [19] and sensitive AuNPs, 40-nm-sized particles were chosen. This is in good Scheme I) as can be seen from Figure 2. In the case of carbon NPs, brighter signals were obtained w agreement with Nath et al. [20], who investigated the effect of particle size on the analytical sensitivity oxidized carbon nanoparticles (oCNPs) (Detection Scheme III) than with carbon NPs that we of an optical biosensor for streptavidin–biotin as a model receptor–ligand pair. The authors showed implemented without further treatment (Detection Scheme II). This is most likely a result of high that the sensitivity of the sensor increases by a factor of three as size of Au NPs increases in the tested functionality and hence more antibody bound to oCNPs compared to plain carbon. In fact, FT range of 13–47 nm. measurements revealed in the oxidized particles reactive groups, such as quinone or conjugat AuNPs have been coupled with anti-IL8 using MHA and carbodiimide [21]. MHA self-assembles ketone [17], that are capable of binding with the amino acids of the detection antibody. Furthermo on the Au particle forming a monolayer. Its carboxy group is subsequently activated with EDC elemental analysis data confirmed oxidation and showed increased oxygen values for oCNPs (9 to covalently bind IL8 through the amino groups present in the amino acids (arginine and lysine). compared with CNPs (5%). However, in contrast to AuNPs both types of carbon NPs lead to visib Alternatively, the antibody might only be mixed with the AuNPs and adsorb on the particles via its spots, proving that Detection Scheme scheme III is more sensitive: the detection limit was 15 ng thiol functions (cysteine). This was realized by Wang et al. [22], who coupled 40 nm Au particles to an anti-aflatoxin M1 antibody via adsorption.

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In our study, the adsorption of antibodies on plain Au nanoparticles was not successful and the coupling antibody to Au via MHA led to mixed results with sometimes poor signals (Detection Scheme I) as can be seen from Figure 2. In the case of carbon NPs, brighter signals were obtained with oxidized carbon nanoparticles (oCNPs) (Detection Scheme III) than with carbon NPs that were implemented without further treatment (Detection Scheme II). This is most likely a result of higher functionality and hence more antibody bound to oCNPs compared to plain carbon. In fact, FTIR measurements revealed in the oxidized particles reactive groups, such as quinone or conjugated ketone [17], that are capable of binding with the amino acids of the detection antibody. Furthermore, elemental analysis data confirmed oxidation and showed increased oxygen values for oCNPs (9%) compared with CNPs (5%). However, in contrast to AuNPs both types of carbon NPs lead to visible spots, proving that Detection Scheme scheme III is more sensitive: the detection limit was 15 ng/L and improved by a factor of more than 10 compared to2018, Detection Scheme II. Interestingly, Posthuma-Trumpie et al. [13] stated that7 of adsorption Biosensors 8, x 13 is commonly used for carbon NP conjugation because it retains the specificity of proteins, important and improved by a factor ofand more than 10 compared to easy Detection Scheme II. Interestingly, for antibody-antigen binding, displays a fast and labeling procedure. This Posthumamight be true for Trumpie et al. [13] stated that adsorption is commonly used for carbon NP conjugation because it special types of carbon black but can by no way be generalized as there are plenty of different grades of retains the specificity of proteins, important for antibody-antigen binding, and displays a fast and carbon black commercially available made by different processes sizes and easy labeling procedure. This might be true for special manufacturing types of carbon black but caninbydifferent no way be surface generalized areas. as there are plenty of different grades of carbon black commercially available made by manufacturing processes in different sizes and surface In different this comparison, carbon NPs finally yield 10-fold higherareas. assay sensitivity compared to AuNPs In this comparison, carbon NPs finally yield 10-fold higher assay sensitivity compared to AuNPs labels. The fact that carbon NPs used as labels can be more sensitive than gold NPs was already labels. The fact that carbon NPs used as labels can be more sensitive than gold NPs was already reported by Gordon et al. [23], who have undertaken a comprehensive survey of literature in PubMed. reported by Gordon et al. [23], who have undertaken a comprehensive survey of literature in Thereby, sensitivities in the low pico-molar range were observed for carbon NPs, even by visual PubMed. Thereby, sensitivities in the low pico-molar range were observed for carbon NPs, even by inspection. have demonstrated that this only thethe case when NPshave have been visualHowever, inspection. we However, we have demonstrated thatis this is only case whencarbon carbon NPs previously by treated nitric acid (oCNPs, Format (III)).(III)). beentreated previously by nitric acid (oCNPs, Format

Figure 2. JPEG images of colorimetric subarrays after incubation with different labels based on

Figure Formats 2. JPEG images colorimetric after with different based on (I)–(VI). Theof corresponding IL8subarrays concentrations are incubation indicated in the columns as 15, labels 135, 1215, Formats10,931, (I)–(VI). The corresponding IL8 concentrations are indicated in the columns as 15, 135, 1215, and 98,376 ng/L. 10,931, and 98,376 ng/L. 3.3. Assay Schemes with Enzymatic and Nanoparticles Labeled Avidin 3.3.1. Evaluation of Enzyme Substrate Three enzyme substrates either based on TMB (from two different providers) or ODI were evaluated using Assay Format (VI). As shown in Figure 3, significant differences in assay performance were observed. As reported by the manufacturer ODI appears as a green precipitate

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3.3. Assay Schemes with Enzymatic and Nanoparticles Labeled Avidin 3.3.1. Evaluation ofxxEnzyme Substrate Biosensors 2018, Biosensors 2018,8, 8, Biosensors 2018, 8, x

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Three enzyme substrates either based on TMB (from two different providers) or ODI were evaluated using Assay Format (VI). As shown in Figure 3, significant differences in assay performance were observed. As reported by the manufacturer ODI appears as a green precipitate after reaction with HRP and normally shows strong adherence on plastic surfaces. TMB precipitates as a dark Biosensors 2018, 8, x 8 of 13blue color and is especially suitable for membrane surfaces, such as nitrocellulose.

Figure 3.3.Mean readouts for IL8 using the Assay Format (VI) with Figure 3. readouts formarker marker IL8 using theusing enzymatic Format (VI) in combination with Figure Mean readouts for marker IL8 using theenzymatic enzymatic Assay Format (VI) in combination withwith 3.Mean Mean readouts for marker IL8 the Assay enzymatic Assay Format (VI) in combination Figure 3. Figure Mean readouts for marker IL8 using the enzymatic Assay Format (VI)in incombination combination different substrates: TMB1 ( ), TMB2 ( ), and ODI ( ). The blank (0 ng/L) is represented by withdifferent different substrates: TMB1 ( ), TMB2 ( ), and ODI ( ). The blank (0 ng/L) is substrates: TMB1TMB1 ( (( (0 isisrepresented by TMB2 and ODI ( The).blank blank (0ng/L) ng/L) represented by by different substrates: (),),TMB2 ), TMB2 (),),and ),ODI and (ODI ().).The The blank (0 ng/L) is represented 1 ng/L on the log scale. represented bythe 1on ng/L on log scale. 11ng/L on log ng/L on the log scale. 1 ng/L thescale. logthe scale. Using the two different TMB substrates, the sensitivities, as defined by the LOD, were different

Using the substrates, the as by the LOD, were different by aUsing factor of 15.different The lowest detectable IL8 concentration 15 as ng/L with TMB1 and ng/L with Using thetwo two different TMB substrates, thesensitivities, sensitivities, asdefined defined by405 the LOD, were different the two different TMB substrates, thewas sensitivities, as by defined by the LOD, were different Using the two different TMBTMB substrates, the sensitivities, defined the LOD, were different TMB2. This suggests that there are different qualities of enzyme substrate available in the market by a factor of 15. The lowest detectable IL8 concentration was 15 ng/L with TMB1 and 405 ng/L with aby factor 15. The lowest detectable IL8 concentration was 15 ng/L with TMB1 andng/L 405 ng/L withwith a factor oflowest 15. The lowest detectable IL8 concentration was 15 ng/L with TMB1 and 405 ng/L by a by factor of 15.ofThe detectable IL8 concentration was 15 ng/L with TMB1 and 405 with which all induce different reaction rates depending on the TMB and H2O2 concentration, pH and TMB2. This suggests that are qualities of substrate available in the TMB2. ThisThis suggests that there there are different different qualities of enzyme enzyme substrate available inmarket the market TMB2. that aremight different of substrate enzyme substrate available inmarket the market TMB2. This suggests that there are there different qualities of enzyme available in the buffer system. suggests Further influencing factors be the qualities solubility product of the precipitating agent which all induce different reaction rates depending on the TMB and H 2 O 2 concentration, pH which all induce different reaction rates depending on the TMB and H 2 O 2 concentration, pH and allTMB induce reaction rates depending on membrane. the H2O2 concentration, pH and with the cationdifferent and the adhesion the precipitate theTMB chip which allwhich induce different reaction rates of depending ononthe andTMB H2 Oand pH andand 2 concentration, buffer system. Further influencing factors be the product of the agent Data reproducibility as reflected by themight small standard deviations was good with all tested buffer system. Further influencing factors might besolubility the solubility product ofprecipitating the precipitating agent buffer system. Further influencing factors besolubility the solubility product ofprecipitating the precipitating agent buffer system. Further influencing factors might bemight the product of the agent substrates which points to a adhesion homogeneous staining pattern and very even distribution of precipitate with the TMB cation and the of the precipitate on the chip membrane. with the TMB cation and the adhesion of the precipitate on the chip membrane. with the TMB cation and the adhesion of the precipitate on the chip membrane. with the TMB cation and the adhesion of the precipitate on the chip membrane. within the spots. The enzyme substrate ODI was not suitable for this kind of assay, as no calibration reproducibility as by small standard deviations good with all Data reproducibility as reflected reflected by the the small standard deviations was was good with all tested tested Data as reflected by in the small standard deviations good all tested DataData reproducibility reflected byis the small standard deviations waswas with all with tested curve was reproducibility obtained.asOur experience actually good agreement with Josephy etgood al. [24] who substrates which points to a homogeneous staining pattern and very even distribution of precipitate substrates which points to a homogeneous staining pattern and very even distribution of precipitate substrates which points to a homogeneous staining pattern and very even distribution of precipitate poor signal-to-noise ratio for colorimetric using ODIvery as theeven enzyme substrate arguing substrates reported which points to a homogeneous stainingassays pattern and distribution of precipitate within the spots. The substrate was not suitable for of as no that or spots. more ofenzyme the dianisidine products formed addition, the observed within theone spots. The enzyme substrate ODI was notunstable. suitable forthis this kind ofassay, assay, ascalibration no calibration within the The enzyme substrate ODI was not suitable for kind this kind of ascalibration no calibration within the spots. The enzyme substrate ODIODI was not are suitable forInthis kind ofauthors assay, asassay, no that ODI is short-lived and disappears within a few minutes [24]. This was not the case in our curve was obtained. Our experience is actually in good agreement with Josephy et al. who was was obtained. OurOur experience is actually in good agreement with Josephy et reported al.et[24] [24] whowho curve obtained. experience actually in good agreement Josephy al. [24] curvecurve was obtained. Our experience is actually inisgood agreement with Josephy etwith al. [24] who experiments. reported poor signal-to-noise ratio for colorimetric assays using ODI as the enzyme substrate arguing reported poor signal-to-noise ratio for colorimetric assays using ODI as the enzyme substrate arguing reported poor signal-to-noise ratio for colorimetric assays using ODI as the enzyme substrate poor signal-to-noise ratio for colorimetric assays using ODI as the enzyme arguing that one arguing In conclusion, TMB1 performed best among the tested enzyme substratessubstrate and was therefore that one or more of the dianisidine products formed are unstable. In addition, the authors observed that one or more of the dianisidine products formed are unstable. In addition, the authors observed that or moreforof theindianisidine products formed are unstable. In addition, the authors used as substrate HRP allformed further investigations. or more of theone dianisidine products are unstable. In addition, the authors observed that ODI isobserved that ODI isisdisappears short-lived and disappears within aaThis few minutes [24]. This was not in that that ODI short-lived and and disappears within few minutes [24]. This was not the the case casecase in our our ODI is short-lived disappears within a was few minutes [24]. This was not the in our short-lived and within a few minutes [24]. not the case in our experiments. 3.3.2. Detection Schemes Using the Streptavidin–Biotin Bridge experiments. experiments. experiments. In conclusion, TMB1 performed best among the tested enzyme substrates and was therefore used Forconclusion, detection of IL8, samples spikedbest withbest IL8 were incubated the chip and the binding waswas In TMB1 performed among the enzyme substrates and therefore In conclusion, conclusion, TMB1 performed best among the tested tested enzyme substrates and was was therefore In TMB1 performed among the with tested enzyme substrates and therefore as substrate for HRP in all further investigations. detected with biotinylated antibodies and made visible in an additional step using Formats (IV)–(VI). used as for in investigations. usedused assubstrate substrate forHRP HRP inall allinfurther further investigations. as substrate for HRP all further investigations. To enhance the colorimetric signal of the gold NPs, the Au was stained with silver. 3.3.2. DetectionThe Schemes Using the Streptavidin–Biotin Bridge enzymatic Format (VI) represents by far the most sensitive approach, with an at least 273.3.2. Detection Schemes Using the 3.3.2.3.3.2. Detection Schemes Using theStreptavidin–Biotin Streptavidin–Biotin Bridge Using the Streptavidin–Biotin fold Detection lower LOD Schemes Assay Formats and (V).Bridge The Bridge good assay performance of the For detection of IL8,compared samplestospiked with IL8(IV) were incubated with the chip and the binding was enzymatic approach (VI) is demonstrated in Figure 2, while spots visible with the naked eye are detection of IL8, samples spiked with IL8 were incubated with the chip and binding was For detection of IL8, samples spiked with IL8 were incubated withwith the chip andthe the(IV)–(VI). binding was was detected For with biotinylated antibodies and made visible in an additional using For detection of IL8, samples spiked with IL8 were incubated theFormats chip and the binding generated at much higher concentrations using Detection Schemes IV and V.step detected with biotinylated antibodies and made visible in an additional step using Formats (IV)–(VI). detected with biotinylated antibodies and made visible in an additional step using Formats (IV)–(VI). To enhance theUsing colorimetric signal the streptavidin–Au gold NPs, thewas Au was stained with detected with biotinylated antibodies and made visible in an additional step using Formats (IV)–(VI). Assay Format (V), of bound further enhanced with silver. silver. Incubation time was 10colorimetric min, colorimetric which ledrepresents to 5-fold signals compared those without silver staining. As 27-fold To enhance the signal of the NPs, the Au stained with silver. To enhance the colorimetric signal ofbrighter the gold NPs, thesensitive Au was stained with silver. The enzymatic Format (VI) by far the most approach, with ansilver. at least To enhance the signal ofgold the gold NPs, thetowas Au was stained with alsoenzymatic reported by others, incubation time is critical: after 10 min, most silver was deposited; after 25an The Format (VI) represents by far the most sensitive approach, with least 27The enzymatic Format (VI) represents by far the most sensitive approach, with an at atan least 27- 27lower LOD compared to Assay Formats (IV) and (V). The good assay performance of the enzymatic The enzymatic Format (VI) represents by far the most sensitive approach, with at least fold lower LOD compared to Assay Formats (IV) and (V). The good assay performance of the foldfold lower LODLOD compared to Assay Formats (IV) (IV) and and (V). (V). The The goodgood assay performance of the lower compared to Assay Formats assay performance of the enzymatic approach (VI) isis demonstrated in 2,2, while spots visible with the eye enzymatic approach (VI) (VI) demonstrated in Figure Figure while spots visible withwith the naked naked eye are are are enzymatic approach is demonstrated in Figure 2, while spots visible the naked eye generated at higher concentrations using Detection Schemes IV generated atmuch much higher concentrations using Detection Schemes IVand and V. V. generated at much higher concentrations using Detection Schemes IV V. and Using Assay Format (V), streptavidin–Au was enhanced with silver. Incubation Using Assay Format (V), bound bound streptavidin–Au was further further enhanced withwith silver. Incubation Using Assay Format (V), bound streptavidin–Au was further enhanced silver. Incubation time was which led 5-fold brighter signals compared to without silver staining. As timetime was 10 10 min, min, which led to to 5-fold brighter signals compared to those those without silver staining. As As was 10 min, which led to 5-fold brighter signals compared to those without silver staining. also reported by others, incubation time is critical: after 10 min, most silver was deposited; after 25 also also reported by others, incubation time is critical: after 10 min, most silver was deposited; after 25 25 reported by others, incubation time is critical: after 10 min, most silver was deposited; after

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approach (VI) is demonstrated in Figure 2, while spots visible with the naked eye are generated at much higher concentrations using Detection Schemes IV and V. Using Assay Format (V), bound streptavidin–Au was further enhanced with silver. Incubation time was 10 min, which led to 5-fold brighter signals compared to those without silver staining. As also reported others, incubation time is critical: after 10 min, most silver was deposited; after 2513min, Biosensorsby 2018, 8, x 9 of the silver enhancement reaction was in saturation [25]. Optimum incubation times reported are in the min,ofthe silver enhancement range 10–15 min [26,27]. reaction was in saturation [25]. Optimum incubation times reported are in the range of 10–15 min [26,27].

3.4. Comparison of the Six Protein Array Assay Schemes 3.4. Comparison of the Six Protein Array Assay Schemes

To identify the assay schemes that are both rapid and accurate, we compared the times required identify the the assay that areassay both rapid and accurate, we compared the times required to fullyTo complete assayschemes as well as the sensitivity, as summarized in Figures 2 and 4. Assay to fully complete the assay as well as the assay sensitivity, as summarized in Figures 2 and 4. Assay Schemes (II) and (III) require three steps in total (spotting of the probes, incubation with the sample, Schemes (II) and (III) require three steps in total (spotting of the probes, incubation with the sample, and incubation with the labeled detection antibody) and represent the schemes with the shortest assay and incubation with the labeled detection antibody) and represent the schemes with the shortest time (4 h). They are certainly the biomarker assays of choice in time-critical medical situations. Clearly, assay time (4 h). They are certainly the biomarker assays of choice in time-critical medical situations. theClearly, assay time not optimized, incubation of Steps 2–4of might further the was assay time was not especially optimized,the especially the times incubation times Stepsbe2–4 mightreduced. be In addition, Format (III) leads to the lowest LOD being 15 ng/L IL8, as low as Assay Format further reduced. In addition, Format (III) leads to the lowest LOD being 15 ng/L IL8, as low as (VI). AssayFrom Figure 2, it(VI). is obvious that those schemes wereassay the only ones were that could detect Format From Figure 2, it isassay obvious that those schemes the only onesconcentrations that could lower than 1215 ng/L IL8. Nevertheless, concentration-dependent black spots were obtained for all detect concentrations lower than 1215 ng/L IL8. Nevertheless, concentration-dependent black spots tested schemes Assay Scheme (I) for (theAssay Au nanoparticle-labeled detection antibody). wereassay obtained for allexcept tested for assay schemes except Scheme (I) (the Au nanoparticle-labeled detection Resultsantibody). indicate that the choice of detection label strongly influences the assay sensitivity and Results range indicate the choice of detection label influencesmade the assay and of measurement of that the immunoassay. This adds to strongly the observations withsensitivity different types measurement range of the immunoassay. This adds to the observations made[28] withordifferent types of nitrocellulose platforms (different modifications and different porosities) generally different nitrocellulose platforms (different modifications and different porosities) [28] or generally different types of surface materials [29] and enzyme substrates. Choosing the appropriate label allows the assay types of to surface materials and enzyme Choosing the appropriate label allows the sensitivity be tuned within[29] a range that bestsubstrates. fits the detection requirements, for example, to monitor assay sensitivity to be tuned within a range that best fits the detection requirements, for example, to the biomarker over several stages of the disease. In general, no clear trend towards the directly labeled monitor the biomarker over several stages of the disease. In general, no clear trend towards the detection antibody or the biotinylated detection antibody was observed. It is the label and its coupling directly labeled detection antibody or the biotinylated detection antibody was observed. It is the label efficiency, not the assay scheme, that governs assay sensitivity. and its coupling efficiency, not the assay scheme, that governs assay sensitivity.

Figure Schematicpresentation presentation of the (Incubation Steps 3, 4,3,and 5) for Figure 4. 4.Schematic the detection detectionofofIL8 IL8binding binding (Incubation Steps 4, and 5) for tested Assay Formats (I)–(VI) including the limit of detection (LOD) (ng/L), dynamic range (ng/L), tested Assay Formats (I)–(VI) including limit of detection (LOD) (ng/L), dynamic range (ng/L), coefficient variation(%). (%).** visually visually determined. andand coefficient ofofvariation determined.

Spot morphology is consistent for all assay schemes as indicated by the uniform size Spot morphology is consistent for3 all schemes indicated by the(15 uniform sizewas (90–120 µm) and (90–120 µm) and appearance of the × 3assay replicate spots.asThe lowest LOD ng/L IL8) observed appearance the 3 ×(III). 3 replicate spots. The lowest LOD (15 ng/L IL8) was observedfor for the Assay Format (III). for AssayofFormat Despite excellent sensitivity, a high BG was calculated enzymatic Despite excellent sensitivity, a high BG was calculated for the enzymatic approach (Format (VI)), about approach (Format (VI)), about 3.5-fold higher compared to the oCNPs assay (Format (III)). However, the background signal stays constant in Formats (III) and (VI). The high BG is likely due to nonspecific precipitation of the colored product and has already been previously reported [30]. To overcome this problem, Liu et al. [30] established a washing strategy based on a ring-oven technique integrated on a paper-based immunodevice to measure carcinoembryonic antigen (CEA). By this

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3.5-fold higher compared to the oCNPs assay (Format (III)). However, the background signal stays constant in Formats (III) and (VI). The high BG is likely due to non-specific precipitation of the colored product and has already been previously reported [30]. To overcome this problem, Liu et al. [30] established a washing strategy based on a ring-oven technique integrated on a paper-based immunodevice to measure carcinoembryonic antigen (CEA). By this method, the authors were able to effectively reduce non-specific binding and improve LOD 10-fold. However, this method requires also additional instrumentation and heating up to 80 ◦ C, which might compromise antibody activity if not properly controlled at the detection zone. Lönnberg and Carlsson [31] compared the detection ability of using colloidal carbon NPs (at the size of 51 nm) as an antibody label to those obtained with enzymatic labels. A nitrocellulose membrane-based immunochromatographic device and a flatbed scanner as a quantitative test system were used. The scanner detected 0.2–170 amol carbon black/mm2 with a coefficient of variation lower than 2% and an LOD of 0.02 amol/mm2 . The detection ability of pure carbon was comparable to the method using horseradish peroxidase (HRP) together with TMB substrate yielding a colored signal of 5 amol HRP/microtiter well. These values are well comparable and about 2 orders of magnitude lower than those of, e.g. polystyrene particles [19]. In contrast to our study, the carbon NPs were adsorbed on the antibodies, because such a process is completed after only 30 min of incubation. In another article by van Dam et al. [32], the sensitivity of carbon NPs conjugates (prepared by adsorption) to detect Schistosomiasis was reported to be equal to that of the corresponding enzyme linked immunosorbent assay (ELISA). Using the oCNPs (III) assay sensitivity was enhanced by 27 times compared to Scheme (V) using strp–AuNPs (V). This fits well with the results of Linares et al. [33] who demonstrated that carbon black had a remarkably low LOD of 0.01 g/L compared to 0.1 g/L and 1 g/L for silver-coated AuNPs and AuNPs, respectively. 3.5. Multiplexed Detection of IL8, VEGF and DCN in Assay Buffer and Synthetic Urine (1:3) Based on the outcome of Sections 3.1–3.4 Assay Scheme (III) using oCNPs and Assay Scheme (VI) using HRP as a label in combination with TMB1 as substrate were further evaluated in a multiplexed chip format. Biomarkers IL8, VEGF, and DCN that were previously confirmed as important markers for detection of recurrence of bladder cancer [1] were spiked into assay buffer and synthetic urine (1:3) and incubated with the chip. The assay format for the three markers was the same as that described for IL8 in Section 2. The respective calibration curves are presented in Figure 5. As is clear, the dynamic range of the assays is—despite the different signal levels—similar in buffer and in urine (1:3), and interferences by urine (1:3), such as low pH, urea, and other inhibitory components, are negligible. Measurement in pure urine, however, did indeed negatively affect assay sensitivity, and, depending on the protein biomarker, the limit of detection was enhanced by more than 10 times. Matrix effects from urine are reported to be differently pronounced for different assay markers. For example, Goodison et al. [34] evaluated a protein biomarker panel (IL8, MMP9, MMP10, ANG, APOE, SDC1, A1AT, PAI1, CA9, and VEGFA) in voided urine samples using a custom multiplex ELISA assay and divided the biomarkers into two sets, one to be measured with 4-fold diluted and one with 200-fold diluted urine samples. Data reproducibility as defined by the coefficient of variation (CV) was comparable for biomarker analysis in assay buffer and urine (1:3) and as low as 5 to 9%. Clearly, dilution of the urinary samples allows highly sensitive biomarker detection, but at the same time runs the risk to miss extremely low biomarker concentrations. Cut-off values reported in literature for IL8 and VEGF in urine were 25–35 ng/L, and 860 ng/L, respectively [3]. No cut-off value for DCN was found. Such low levels suggest that concentrators or signal enhancement kits shall be used to further improve assay sensitivity and meet the requirements for protein biomarker detection in therapy monitoring. Alternatively, several antibodies and antibody pairs might be tested in terms of sensitivity, specificity, physical properties, and recognition of native protein, and the best one selected for implementation in the multiplex chip [3].

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Figure 5. Normalized colorimetric signals in (a) assay buffer with labels oCNPs (Assay Scheme III); Figure 5.5.Normalized colorimetric signals in assay buffer with labels oCNPs (Assay Scheme III); Figure Normalized colorimetric signals in(a) (a)in assay buffer with labels oCNPs (Assay Scheme III); III); 5. Normalized colorimetric signals (a) assay buffer with labels oCNPs (Assay Scheme Figure 5.Figure Normalized colorimetric signals in (a) assay buffer with labels oCNPs (Assay Scheme III); (b) in urine (1:3) with oCNPs (c) in assay buffer using HRP (Assay Scheme VI); (d) in urine (1:3) with inin urine (1:3) with oCNPs (c) inin(c) assay buffer using HRP (Assay Scheme inin(d) urine (1:3) with (b) in urine (1:3) with oCNPs in assay buffer using HRP (Assay Scheme VI); in urine (1:3) (b)urine urine (1:3) with oCNPs (c)assay assay buffer using HRP (Assay Scheme VI); (d) urine (1:3) withwith (b)(b) in (1:3) with oCNPs (c) in buffer using HRP (Assay Scheme VI);VI); (d)(d) in urine (1:3) with HRP. Calibration curves for 0 to 295 µg/L:  IL8,  VEGF, and  DCN; including standard VEGF, and including standard HRP. Calibration curves 0 0to 295 HRP. Calibration curves 0toto 295µg/L: µg/L: IL8, and DCN; including standard deviations IL8, IL8, VEGF, VEGF, and DCN; DCN; including standard HRP. Calibration curves for 295 µg/L:  IL8,  VEGF, and  DCN; including standard HRP. Calibration curves forfor 0 for to 295 µg/L: deviations of three replicates. The blank signal (0 ng/L) is represented by 15 ng/L on the log scale. deviations ofofthree replicates. The blank signal (0(0ng/L) isisrepresented on the log of three replicates. TheThe blank signal (0 ng/L) is represented byby 15by ng/L on the log scale. deviations three replicates. The blank signal ng/L) represented by15 15ng/L ng/L on the logscale. scale. deviations of three replicates. blank signal (0 ng/L) is represented 15 ng/L on the log scale.

4. Conclusions

4.4.Conclusions Conclusions 4. Conclusions 4. Conclusions

Six protein array assay schemes and three different labels (Au, carbon, enzyme) were evaluated

protein array assay schemes and three different labels (Au, carbon, enzyme) were evaluated Six protein array assay schemes and three different labels (Au, carbon, enzyme) were evaluated SixSix protein array assay schemes and three different labels (Au, carbon, enzyme) were evaluated Six protein array assay schemes and three different labels (Au, carbon, enzyme) were evaluated to identify the most sensitive colorimetric assay scheme to be exploited in an on-chip immunoassay the most sensitive colorimetric assay scheme exploited in an on-chip immunoassay toidentify identify the most sensitive colorimetric assay scheme tobe be exploited inin an on-chip immunoassay for IL8, DCN, and VEGF, three assay protein markers that have beeninpreviously demonstrated to be for to to identify the most sensitive colorimetric scheme to to be exploited an on-chip immunoassay to identify the most sensitive colorimetric assay scheme to be exploited an on-chip immunoassay important for thethree detection of bladder cancer recurrence. The highest signal intensitytoand assay IL8, DCN, and VEGF, three protein markers that have been previously demonstrated for IL8, DCN, and VEGF, three protein markers that have been previously demonstrated tobe be forfor IL8, DCN, and VEGF, protein markers have been previously demonstrated to to be DCN, and VEGF, three protein markers that have been previously demonstrated be important sensitivity was observed for the enzymatic Assay Format (VI) with TMB1 as an enzyme substrate and important for the detection of bladder cancer recurrence. The highest signal intensity and assay important for the detection of bladder cancer recurrence. The highest signal intensity and assay important for detection the detection of bladder recurrence. The highest signal intensity andsensitivity assay for the of bladder cancercancer recurrence. The highest signal intensity and assay was Format (III) using oxidized carbon NPs (oCNPs). Herein, we could show that those schemes perform sensitivity was observed the enzymatic Assay Format (VI) with as substrate and sensitivity was observed for the enzymatic Assay Format (VI) with TMB1 asan anenzyme enzyme substrate and (III) sensitivity was observed forfor the enzymatic Assay Format (VI) with TMB1 as an enzyme substrate and observed for the enzymatic Assay Format (VI) with TMB1 asTMB1 an enzyme substrate and Format equally well in synthetic urine (1:3) and assay buffer in a multiplexed arrangement. With this Format (III) using oxidized carbon NPs (oCNPs). Herein, could show that those schemes perform Format (III) using oxidized carbon NPs (oCNPs). Herein, we could show that those schemes perform Format (III) using oxidized carbon NPs (oCNPs). Herein, wewe could show that those schemes perform using oxidized carbon NPs (oCNPs). Herein, we could show that those schemes perform equally well colorimetric chip, a cost-effective, fast (4 h), simple, and efficient tool was established, which has high equally well in synthetic urine (1:3) and assay buffer in a multiplexed arrangement. With this equally well in urine synthetic urine (1:3) and assay buffer inmultiplexed a arrangement. multiplexed arrangement. With thischip, equally in synthetic urine (1:3) and assay buffer in a arrangement. With this inwell synthetic (1:3) and assay buffer in a multiplexed With this colorimetric potential for use in doctors’ offices as a non-invasive tool in therapy monitoring. colorimetric chip, aacost-effective, fast (4(4h), simple, and efficient tool was established, which high colorimetric chip, cost-effective, fast h), simple, and efficient tool was established, which has high colorimetric chip, a cost-effective, fast (4 and h), simple, and efficient tool was established, which hashas high a cost-effective, fast (4 h), simple, efficient tool was established, which has high potential for use in potential for use in doctors’ offices as a non-invasive tool in therapy monitoring. potential for use in doctors’ offices as a non-invasive tool in therapy monitoring. Acknowledgments: The authors thank the European Commission for financial support within FP7 Dipromon potential for useoffices in doctors’ offices as a non-invasive toolmonitoring. in therapy monitoring. doctors’ as a non-invasive tool in therapy (development of an integrated protein- and cell-based device for noninvasive diagnostics in the urogenital tract, no. 306157). Acknowledgments: The authors thank the European Commission for financial support within FP7 Dipromon Acknowledgments: The authors thank the Commission support within FP7 Dipromon Acknowledgments: The authors thank theEuropean European Commission forfinancial financial support within FP7 Dipromon Acknowledgments: The authors thank the European Commission forfor financial support within FP7 Dipromon (development of an integrated proteinand cell-based device for noninvasive diagnostics in the urogenital (development of an integrated proteinand cell-based device for noninvasive diagnostics in the urogenital tract, (development of an integrated proteinand cell-based device for noninvasive diagnostics in the urogenital tract,tract, (developmentAuthor of an integrated proteincell-based device for diagnostics in the urogenital tract, Contributions: C.P. and conceived and designed the noninvasive experiments; S.G. and S.D. performed the experiments; no. 306157). no. 306157). no. 306157). S.G. and U.S. analyzed the data; C.P. and S.G. wrote the paper. no. 306157). Author Contributions: C.P. conceived and designed the experiments; S.G. and S.D. performed the experiments; Conflicts of Interest: The authors declare no conflict of paper. interest. Author Contributions: C.P. conceived and designed the S.G. and S.D. performed Author Contributions: C.P. conceived and designed theexperiments; experiments; S.G. and S.D. performed theexperiments; experiments; Author Contributions: C.P. conceived and designed the experiments; S.G. and S.D. performed thethe experiments; S.G. and U.S. analyzed the data; C.P. and S.G. wrote the S.G. and U.S. analyzed C.P. and S.G. wrote S.G. and U.S. analyzed thedata; data; C.P. and S.G. wrote thepaper. paper. S.G. and U.S. analyzed thethe data; C.P. and S.G. wrote thethe paper. Conflicts of Interest: The authors declare no conflict of interest. Conflicts The authors declare ofofinterest. Conflicts ofInterest: Interest: The authors declare noconflict conflict interest. Conflicts of of Interest: The authors declare nono conflict of interest.

References 1. 2.

Budman, L.I.; Kassouf, W.; Steinberg, J.R. Biomarkers for detection and surveillance of bladder cancer. Can. Urol. Assoc. J. 2008, 2, 212–221. [CrossRef] [PubMed] Tilki, D.; Burger, M.; Dalbagni, G.; Grossman, H.B.; Hakenberg, O.W.; Palou, J.; Reich, O.; Rouprêt, M.; Shariat, S.F.; Zlotta, A.R. Urine markers for detection and surveillance of non-muscle-invasive bladder cancer. Eur. Urol. 2011, 60, 484–492. [CrossRef] [PubMed]

Biosensors 2018, 8, 10

3. 4.

5. 6.

7. 8. 9. 10. 11.

12. 13.

14.

15.

16.

17. 18.

19. 20.

21.

12 of 13

Gogalic, S.; Sauer, U.; Doppler, S.; Preininger, C. Bladder cancer biomarker array to detect aberrant levels of proteins in urine. Analyst 2015, 140, 724–735. [CrossRef] [PubMed] Gogalic, S.; Sauer, U.; Doppler, S.; Heinzel, A.; Perco, P.; Lukas, A.; Simpson, G.; Pandha, H.; Horvath, A.; Preininger, C. Validation of a protein panel for the non-invasive detection of recurrent non-muscle invasive bladder cancer. Biomarkers 2017, 19, 1–8. [CrossRef] [PubMed] Satija, J.; Punjabi, N.; Mishraac, D.; Mukherji, S. Plasmonic-ELISA: Expanding horizons. RSC Adv. 2016, 6, 85440–85456. [CrossRef] Lan, J.; Xu, W.; Wan, Q.; Zhang, X.; Lin, J.; Chen, J.; Chen, J. Colorimetric determination of sarcosine in urine samples of prostatic carcinoma by mimic enzyme palladium nanoparticles. Anal. Chim. Acta 2014, 825, 63–68. [CrossRef] [PubMed] Wei, W.; Zhang, C.; Qian, J.; Liu, S. Multianalyte immunoassay chip for detection of tumor markers by chemiluminescent and colorimetric methods. Anal. Bioanal. Chem. 2011, 401, 3269–3274. [CrossRef] [PubMed] Geckeler, K.E.; Eckstein, H. (Eds.) Bioanalytische und Biochemische Labormethoden; Vieweg & Sohn Verlagsgesellschaft mbH: Wiesbaden, Germany, 2008. Penn, S.G.; He, L.; Natan, M.J. Nanoparticles for bioanalysis. Curr. Opin. Chem. Biol. 2003, 7, 609–615. [CrossRef] [PubMed] Ranjan, R.; Esimbekova, E.N.; Kratasyuk, V.A. Rapid biosensing tools for cancer biomarkers. Biosens. Bioelectron. 2017, 87, 918–930. [CrossRef] [PubMed] Jiang, L.; Yu, Z.; Du, W.; Tang, Z.; Jiang, T.; Zhang, C.; Lu, Z. Development of a fluorescent and colorimetric detection methods-based protein microarray for serodiagnosis of TORCH infections. Biosens. Bioelectron. 2008, 24, 376–382. [CrossRef] [PubMed] Goryacheva, I.Y.; Lenain, P.; De Saeger, S. Nanosized labels for rapid immunotests. TrAC Trends Anal. Chem. 2013, 46, 30–43. [CrossRef] Posthuma-Trumpie, G.A.; Wichers, J.H.; Koets, M.; Berendsen, L.B.J.M.; Amerongen, A. Amorphous carbon nanoparticles: A versatile label for rapid diagnostic (immuno)assays. Anal. Bioanal. Chem. 2011, 402, 593–600. [CrossRef] [PubMed] Van Amerongen, A.; Wichers, J.H.; Berendsen, L.B.; Timmermans, A.J.; Keizer, G.D.; van Doorn, A.W.; Bantjes, A.; van Gelder, W.M. Colloidal carbon particles as a new label for rapid immunochemical test methods: Quantitative computer image analysis of results. J. Biotechnol. 1993, 30, 185–195. [CrossRef] Noguera, P.S.; Posthuma-Trumpie, G.A.; van Tuil, M.; van der Wal, F.J.; Boer, A.D.; Moers, A.P.H.A.; van Amerongen, A. Carbon nanoparticles as detection labels in antibody microarrays. Detection of genes encoding virulence factors in Shiga toxin-producing Escherichia coli. Anal. Chem. 2011, 83, 8531–8536. [CrossRef] [PubMed] Lavorgna, M.; Romeoa, V.; Martonea, A.; Zarrellia, M.; Giordanoa, M.; Buonocorea, G.G.; Qub, M.Z.; Feic, G.X.; Xiac, H.S. Silanization and silica enrichment of multiwalled carbon nanotubes: Synergistic effects on the thermal-mechanical properties of epoxy nanocomposites. Eur. Polym. J. 2013, 49, 428–438. [CrossRef] Mattsson, L.; Jungmann, C.; Lieberzeit, P.A.; Preininger, C. Modified carbon black as label in a colorimetric on-chip immunoassay for histamine. Sens. Actuators B 2017, 246, 1092–1099. [CrossRef] Noguera, P.; Posthuma-Trumpie, G.A.; van Tuil, M.; van der Wal, F.J.; de Boer, A.; Moers, A.P.H.A.; van Amerongen, A. Carbon nanoparticles in lateral flow methods to detect genes encoding virulence factors of Shiga toxin-producing Escherichia coli. Anal. Bioanal. Chem. 2011, 399, 831–838. [CrossRef] [PubMed] Seydack, M. Nanoparticle labels in immunosensing using optical detection methods. Biosens. Bioelectron. 2005, 20, 2454–2469. [CrossRef] [PubMed] Nath, N.; Chilkoti, A. A colorimetric gold nanoparticle biosensor: Effect of particle size on sensitivity. In Proceedings of the Second Joint 24th Annual Conference and the Annual Fall Meeting of the Biomedical Engineering Society EMBS/BMES Conference, Houston, TX, USA, 23–26 October 2002; Volume 1, pp. 574–575. Shi, H.; Yuan, L.; Wu, Y.; Liu, S. Colorimetric immunosensing via protein functionalized gold nanoparticle probe combined with atom transfer radical polymerization. Biosens. Bioelectr. 2011, 26, 3788–3793. [CrossRef] [PubMed]

Biosensors 2018, 8, 10

22.

23. 24.

25. 26. 27.

28. 29.

30.

31. 32.

33.

34.

13 of 13

Wang, J.J.; Liu, B.H.; Hsu, Y.T.; Yu, F.Y. Sensitive competitive direct enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip for detecting aflatoxin M1 in milk. Food Control 2011, 22, 964–969. [CrossRef] Gordon, J.; Michel, G. Analytical sensitivity limits for lateral flow immunoassays. Clin. Chem. 2008, 54, 1250–1251. [CrossRef] [PubMed] Josephy, P.D.; Eling, T.; Mason, R.P. The horseradish peroxidase-catalyzed oxidation of 3,5,30 ,50 tetramethylbenzidine. Free radical and charge-transfer complex intermediates. J. Biol. Chem. 1982, 257, 3669–3675. [PubMed] Liang, R.-Q.; Tan, C.-Y.; Ruan, K.-C. Colorimetric detection of protein microarrays based on nanogold probe coupled with silver enhancement. J. Immunol. Methods 2004, 285, 157–163. [CrossRef] [PubMed] Yang, M.; Wang, C. Label-free immunosensor based on gold nanoparticle silver enhancement. Anal. Biochem. 2009, 385, 128–131. [CrossRef] [PubMed] Tang, J.; Xu, Z.; Zhou, L.; Qin, H.; Wang, Y.; Wang, H. Rapid and simultaneous detection of Ureaplasma parvum and Chlamydia trachomatis antibodies based on visual protein microarray using gold nanoparticles and silver enhancement. Diagn. Microbiol. Infect. Dis. 2010, 67, 122–128. [CrossRef] [PubMed] Cretich, C.; Sedini, V.; Damin, F.; Pelliccia, M.; Sola, L.; Chiari, M. Coating of nitrocellulose for colorimetric DNA microarrays. Anal. Biochem. 2010, 397, 84–88. [CrossRef] [PubMed] Le Goff, G.C.; Corgier, B.P.; Mandona, C.A.; De Crozals, G.; Chaix, C.; Loïc, J.; Blum, L.J.; Marquette, C.A. Impact of immobilization support on colorimetric microarrays performances. Biosens. Bioelectr. 2012, 35, 94–100. [CrossRef] [PubMed] Liu, W.; Guo, Y.; Zhao, M.; Li, H.; Zhang, Z. Ring-oven washing technique integrated paper-based immunodevice for sensitive detection of cancer biomarker. Anal. Chem. 2015, 87, 7951–7957. [CrossRef] [PubMed] Lönnberg, M.; Carlsson, J. Quantitative detection in the attomole range for immunochromatographic tests by means of a flatbed scanner. Anal. Biochem. 2001, 293, 224–231. [CrossRef] [PubMed] Van Dam, G.J.; Wichers, J.H.; Ferreira, T.M.F.; Ghati, D.; van Amerongen, A.; Deelder, A.M. Diagnosis of schistosomiasis by reagent strip test for detection of circulating cathodic antigen. J. Clin. Microbiol. 2004, 42, 5458–5461. [CrossRef] [PubMed] Linares, E.M.; Kubota, L.T.; Michaelis, J.; Thalhammer, S. Enhancement of the detection limit for lateral flow immunoassays: Evaluation and comparison of bioconjugates. J. Immunol. Methods 2012, 375, 264–270. [CrossRef] [PubMed] Goodison, S.; Ogawa, O.; Matsui, Y.; Kobayashi, T.; Miyake, M.; Ohnishi, S.; Fujimoto, K.; Dai, Y.; Shimizu, Y.; Tsukikawa, K.; et al. A multiplex urinary immunoassay for bladder cancer detection: Analysis of a Japanese cohort. J. Transl. Med. 2016, 14, 287–295. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).