Signal Amplified Gold Nanoparticles for Cancer Diagnosis on Paper

Dec 28, 2017 - ... KLK3 antigen on chromatography paper were recorded using a smartphone (iPhone, Apple, ..... After 6 min, the color stayed more cons...
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Signal Amplified Gold Nanoparticles for Cancer Diagnosis on Paper-Based Analytical Devices Jia-Yu Huang, Hong-Ting Lin, Tzu-Heng Chen, Chung-An Chen, Huan-Tsung Chang, and Chien-Fu Chen ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00823 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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Signal Amplified Gold Nanoparticles for Cancer Diagnosis on PaperBased Analytical Devices Jia-Yu Huang†, Hong-Ting Lin‡, Tzu-Heng Chen†, Chung-An Chen‡, Huan-Tsung Chang†*, and Chien-Fu Chen‡§* †

Department of Chemistry, National Taiwan University, Taipei 106, Taiwan

‡

Institute of Applied Mechanics, National Taiwan University, Taipei 106, Taiwan

§

Center for Emerging Material and Advanced Devices, National Taiwan University, Taipei 106, Taiwan

KEYWORDS: Signal amplification, cancer diagnosis, gold nanoparticles, colorimetric assay, paper-based analytical device

ABSTRACT: In this work, we report a highly sensitive colorimetric sensing strategy for cancer biomarker diagnosis using gold nanoparticles (AuNPs) labelled with biotinylated poly-adenine ssDNA sequences and streptavidin-horseradish peroxidase for enzymatic signal enhancement. By adopting this DNA-AuNP nanoconjugate sensing strategy, we were able to eliminate the complicated and costly thiol-binding process typically used to modify AuNP surfaces with ssDNA. In addition, different antibodies can be introduced to the AuNP surfaced via electrostatic interactions to provide highly specific recognition sites for biomolecular sensing. Moreover, multiple, simultaneous tests can be rapidly performed with low sample consumption by incorporating these surface-modified AuNPs into a paper-based analytical device that can be read using just a smartphone. As a result of these innovations, we were able to achieve a detection limit of 10 pg/mL for a prostate specific antigen in a test that could be completed in as little as 15 minutes. These results suggest that the proposed paper platform possesses the capability for sensitive, high-throughput, and on-site prognosis in resource-limited settings.

There are no efficient amplification methods for proteins that are analogous to the polymerase chain reaction (PCR) of DNA. In conventional enzyme-based colorimetric immunoassays, a single enzyme molecule, such as horseradish peroxidase (HRP), can oxidize the targeted substrate using hydrogen peroxide to yield a characteristic change that is detectable by spectroscopic methods. However, in these systems a 1:1 ratio of the enzyme and detection antibody is typically used due to steric limitations.1 As a result, it remains a challenge to detect small quantities of protein biomarkers for early stage cancer diagnosis with cost-effective, simple, and highly sensitive and specific diagnostic strategies.2 Many efforts have been directed toward the exploration of novel means to enhance detection signals in order to improve the sensitivity of protein sensing platforms.3-4 To further increase the sensitivity of enzymebased colorimetric immunoassays, a key strategy is to enhance the ratio between the enzyme and the target analyte. To achieve this goal, there are a number of sensing/signal amplification strategies that have been developed using various nanomaterials in biochemical analytical platforms.

Among these materials, gold nanoparticles (AuNPs) have been widely used as labels or carriers of signal molecules for analytical signal amplification.5-7 Because of their narrow size distribution, catalysis, strong biocompatibility, and ease of modification with thiol groups, AuNPs can not only serve as carriers for the immobilization of bioconjugate probes (e.g., antibodies, DNA, and aptamers), but an abundant number of biorecognition elements or optical/electrochemical tags can also be immobilized on their surfaces to provide more binding sites or signal amplification of the target analyte for a single recognition reaction.812 However, the sensing enzymes are typically physically adsorbed to the AuNPs in these applications, resulting in unstable bonding between them. In order to enhance the reproducibility and sensitivity of these AuNP-based tests, researchers have studied stronger binding affinity methods.13 For example, Irudayaraj et al. used biotinylated AuNPs and streptavidin conjugated HRP to develop a highly sensitive lateral-flow assay for E. coli detection.14 Another strategy modified AuNPs with an atom transfer radical polymer to load more enzymes and

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recognition elements of the cancer biomarker Nogo-66 to lower the detection limit by a factor of 81 compared to a standard ELISA test.15 In addition, the signal intensity of the immunogold labeled target analyte can be enhanced through silver staining, a technique in which silver particles are precipitated onto the surface of the AuNPs.16-19 Moreover, in order to achieve both the purification and quantification of analytes, Mirkin et al. developed a biobarcode assay for detecting proteins with PCR-like sensitivity.20-22 In their design, AuNPs were coated with both a polyclonal antibody that was specific for the target protein and hundreds of barcode DNA strands that could be chemically released and amplified via PCR. This bio-barcode strategy effectively translates otherwise difficult to amplify proteins into a convenient PCR method, which has been employed to detect protein markers for early-stage diagnosis of Alzheimer’s disease,21 as well as to monitor the biochemical recurrence of cancer cells after radical prostatectomy.23 However, all of these highly sensitive detection methods require costly thiolated DNA probes for assembly onto the AuNP surfaces through Au−S bonding. In this study, we used high density biotinylated oligonucleotides containing consecutive polyadenine (poly(A)) sequences, which serve as effective anchoring blocks for attachment to the AuNP surfaces. The poly(A) sequences, which feature a collectively high binding affinity to the gold surface, preferentially serve as strong anchoring block to the AuNPs, comparable to that of Au−S chemistry.24-29 Moreover, the DNA-AuNP conjugates prepared with this poly(A) strategy exhibit faster hybridization kinetics compared to thiolated DNA-AuNPs. The assembled poly(A) layer also retains electrostatic and steric repulsions that promote the extension of the oligonucleotides into solution, thus adopting the “stand-up” conformation on the AuNP surfaces, which favors much higher density anchoring.24, 30 The addition of streptavidin-HRP molecules that bind to the biotinylated poly(A) DNA sequences via the biotinstreptavidin interaction provide the opportunity for enhanced signal amplification through the increased enzymatic reaction of antibody-antigen bioconjugation events on the AuNP surfaces. Adding 3,3',5,5'-tetramethylbenzidine (TMB) to this system acts as a hydrogen donor for the reduction of hydrogen peroxide to water by the HRP enzyme. In this reaction, TMB is oxidized to 3,3',5,5'-tetramethylbenzidine diimine, which is blue colored, creating a corresponding color change in solution that can be detected by a spectrophotometer at a wavelength of 650 nm. In this manner, increasing color change of the solution can be quantitatively interpreted as the target analyte amount.

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In order to verify the feasibility of the proposed platform, we chose the prostate-specific antigen (PSA), also known as gamma-semino-protein or kallikrein-3 (KLK3), a glycoprotein enzyme encoded in humans by the KLK3 gene, as a targeted biomarker.31-33 The serum level of KLK3 has been widely used for the clinical diagnosis of prostate cancer, with an abundance of over 4 ng/mL indicating a high probability of the disease. It can also be used as an unambiguous indicator of response to cancer therapy and recurrence for patients who have undergone radical prostatectomy.3435

To achieve a high bonding density of the streptavidinHRP molecules on the AuNPs for increased signal intensity while remaining highly specific to the target analyte provided by monoclonal antibodies attached to the AuNPs surface via electrostatic interactions,36 we studied different orders of AuNP modification, with either the antibody (anti-human IgG (aHIgG) or anti-kallikrein-3 (aKLK3)) or the HRP-poly(A) ssDNA anchored on the nanoparticles first (named Ab-AuNPs and ssDNA/HRP-AuNPs, respectively), followed by colorimetric analysis of the high intensity signal of this protein biomarker assay to verify the highest signal amplification factor with high specificity. In addition, different ratios of streptavidin-HRP to biotinylated oligonucleotide sequences were also studied to achieve the highest density of HRP molecules on a single AuNP at minimal cost. To further avoid the use of sophisticated equipment and minimize the required resources, we combined this AuNP sensing strategy with a paper-based analytical device (PAD) to produce a colorimetric test.37-39 The main advantage of this paper-based platform is due to its cellulose fibers, which allow liquids to penetrate into the hydrophilic matrix without external power sources. Moreover, its ability to easily store analytes or reagents, the lower sample consumption, faster reaction times, enhanced portability, and the potential for simultaneous multiple analyte detection and qualitative analyses via a smartphone make this system relevant for diagnostic applications in resource limited settings.40-43 By monitoring blue/red color ratio changes on defined areas of the PAD containing the HRP-DNA and antibody functionalized AuNPs (i.e., the Au-nanoprobes) upon exposure to the analyte solution, we can semi-quantitatively determine the concentration of target KLK3 molecules in the sample. After optimizing the test, we achieved a 10 pg/mL detection limit for KLK3, with results ready in as little as 15 min. These findings suggest the proposed platform possesses the potential for sensitive and high-throughput on-site prognoses, particularly in resource-limited settings

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where paper-based tests are faster, easier, and cheaper to use.

EXPERIMENTAL SECTION Reagents and Materials. Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4), trisodium citrate, bovine serum albumin (BSA), Tris-Borate-EDTA buffer, sucrose, phosphate buffered saline with Tween-20, phosphate buffered saline, 3,3',5,5'-tetramethylbenzidine liquid (TMB), KLK3 antigen, anti-kallikrein-3 (aKLK3) antibody, and streptavidin-HRP were purchased from Sigma-Aldrich (St. Louis, MO). Biotinylated oligonucleotide sequences were synthesized and purified by Bio Basic Inc. (Bio Basic, Ontario, Canada). Whatman cellulose chromatography paper was obtained from GE Healthcare (1 Chr, Little Chalfont, UK). Ultrapure water (18.2 mΩ·cm) was filtered through a MilliQ system (Millipore, Milford, MA). Instrumentation and Characterization. The size and morphology of the synthesized and modified AuNPs were verified by a transmission electron microscope (TEM; H7100, Hitachi High-Technologies, Tokyo, Japan). The quantity of streptavidin-HRP and biotinylated oligonucleotide sequences (HRP-DNA) anchored to the AuNPs was characterized using a UV−Vis spectrophotometer (Cintra 10e, GBC, Victoria, Australia). The absorbance wavelength of TMB is 650 nm, while the AuNPs is 520 nm.43-44 The red, green, blue (RGB) color values of the detection results of the KLK3 antigen on chromatography paper were recorded using a smartphone (iPhone, Apple, CA) under artificial white light without flash and a USB digital microscope (UPG650, Upmost, Taiwan). All captured images were immediately uploaded to cloud storage (Dropbox, San Francisco, CA), followed by the analysis of the RGB values using a self-developed iPhone app and ImageJ software in parallel (National Institutes of Health, Bethesda, MD). Preparation of the Au-Nanoprobes. A schematic illustration of the Au-nanoprobe preparation process featuring the two different orders of synthesis (i.e., antibody or ssDNA/HRP first) is shown in Figure 1A. For these initial experiments, we used human IgG as the target molecule due to its cost advantages. Citrate-capped AuNPs were synthesized using the citrate-mediated reduction of HAuCl4.45 The concentration of the AuNP colloid was estimated from the absorbance spectrum according to the Beer-Lambert law.46 For the preparation of AuNPs modified with aHIgG antibodies before the addition of poly(A) ssDNA, the antibodies were first mixed with the AuNPs at a molar ratio of 15 and incubated for 20 min at 700 rpm. 100 μL of 1 mg/mL BSA was then added to this solution for blocking nonspecific binding and

subsequently stirred for another 20 min at 700 rpm. Any unbound aHIgG antibodies were removed by centrifugation (16000 rpm for 20 min) at 4 °C, followed by re-suspension of the antibody-AuNP pellet in 300 μL of 2 mM borate buffer at pH 7.4 featuring 10% sucrose. Afterward, the mixture was incubated at room temperature with biotinylated DNA (Poly(A)20 anchoring block) at a molar ratio of 100 biotinylated DNA to AuNPs for 2 h.28 The solution was then added 10 mM sodium phosphate buffer (pH 7.4) with 0.1 M NaCl, allowed to stand for 12 h, and then washed twice in 10 mM sodium phosphate buffer (pH 7.4) with 0.1 M NaCl via centrifugation (12,000 rpm, 20 min, 4℃) to remove excess DNA. The clear supernatant was then carefully removed and the precipitated gold conjugates were re-suspended in phosphate buffer (0.3 M NaCl, 10 mM sodium phosphate, pH 7.4). After that, streptavidin-HRP was added to the gold complex solution in a molar ratio of 100 to AuNPs for 1 h at room temperature. Finally, the Au-nanoprobes were collected by centrifugation and stored at 4 °C until further use. For the preparation of AuNPs modified with poly(A) ssDNA before the addition of the aHIgG antibodies, the procedure remained the same except the order of the antibody and poly(A) ssDNA steps were switched. Optimal molar ratio of streptavidin-HRP to biotinylated-DNA. We also investigated the optimal ratio of streptavidin-HRP molecules to the biotinylated-poly(A) ssDNA anchored to the AuNPs. A schematic illustration of the Au-nanoprobe preparation process is shown in Figure 2A. Different molar ratios of streptavidin-HRP to biotinylated-poly(A) ssDNA anchored on the Au-nanoprobes were tested, including 50:100:1, 75:100:1, and 100:100:1 (streptavidin-HRP : biotinylated-DNA : AuNP). The preparation method was the same as mentioned previously for the ssDNA/HRP-AuNP nanoprobes, except the ratios of streptavidin-HRP molecules and biotinylated-DNA were varied. We estimated the concentration of streptavidin-HRP on the different Au-nanoprobes by measuring the absorbance spectra of the samples after the addition of TMB and comparing those values to calibration curves of standard streptavidin-HRP solutions, including 0.96 nM, 0.48 nM, 0.24 nM, and 0.12 nM, 2 min and 4 min after TMB had been added. The external standard method was used to estimate the actual ratio of streptavidin-HRP anchored to the Aunanoprobes. Signal Enhanced Paper-Based ELISA Test. Using the optimized molar ratio conditions, the ssDNA/HRP-AuNP nanoprobes were synthesized using biotinylated poly(A) ssDNA, streptavidin HRP, and anti-KLK3 labeled AuNPs

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Scheme 1. Schematic illustration of the P-ELISA test utilizing the functionalized gold-nanoprobe sensing strategy.

(Scheme 1). AuNPs were employed as carriers to load the detector antibody and signal amplification reagent. The detection results were obtained using a PAD formed on cellulose paper using the solid-ink printing method.47-49 The detection areas were 3 mm in diameter and fabricated by printing circular hydrophobic barriers with a solid wax printer (Xerox 8570, Fuji, Japan) on the surface of the paper and then heating the material to 120 °C for 10 min to allow the wax to penetrate between the cellulose fibers. In addition, reference colors were simultaneously printed on the PAD to provide a standard for different lighting conditions. In order to verify the efficacy of the proposed signal enhancement method, we chose KLK3 as a model antigen for the paper-based ELISA (P-ELISA) tests. In its simplest form, the protocol for the direct P-ELISA method is as follows: to immobilize KLK3 on the PADs, we added 3 µL of the different concentrations of KLK3 solution, from 0.003 to 1 ng/mL in PBS, to the printed testing zone and allowed it to dry for 5 min under ambient conditions. We then blocked each testing zone by adding 3 µL of a blocking buffer (1% w/v BSA in PBS) and allowed the area to dry for 5 min under ambient conditions. 3 µL of the ssDNA/HRPAuNP nanoprobe solution was then added to each zone and allowed to dry for another 5 min. Each test area was washed with 50 µL of PBS by pipetting and withdrawing the liquid from the testing zone 10 times, and then we changed the micropipette tip and buffer solution and repeating the washing process once more. Finally, 3 µL of TMB was added to the testing spot and the color-producing enzymatic reaction between TMB and HRP was allowed to proceed for 4 min under ambient conditions. We recorded the resulting images of the P-ELISA test using a smartphone under artificial white light without a flash. The results were also confirmed using a USB charge-coupled device (CCD) camera in order to eliminate any variance of the environmental conditions. Images were simultaneously transmitted to cloud storage or USB connection, and

the intensity of the color was measured using ImageJ software. Analysis of KLK3 Samples Spiked in Serum. KLK3 antigen solutions ranging in concentration from 0.3–300 pg/mL spiked in 1/10 diluted human serum extracted and centrifuged from a healthy volunteer (male, 25-years-old) were used to simulate sample testing in a clinical setting. The detection processes were identical to those mentioned previously utilizing the optimized functionalized Au-nanoprobes, followed by measuring the colorimetric results on the PADs and acquiring the data via smartphone or CCD camera for subsequent analysis. The detection results were then recorded and transmitted to a server to instantly receive quantitative data via cloud computing.

RESULTS AND DISCUSSION In this study, we demonstrate a highly sensitive bioassay that can be realized using functionalized AuNPs as carriers on which to load detector antibodies and abundant signal amplification molecules to create a novel colorimetric sensing platform. High density HRP enzymes linked to the AuNPs were prepared via the high binding affinity of poly(A) ssDNA to Au and the biotin-avidin interaction, thus avoiding the use of costly and complex thiolated-labeling molecules. In addition, to make this analytical strategy compatible with resource-constrained environments, the assay was performed on a paper-based platform in order to achieve lower sample and reagent consumption, as well as a shorter assay time. A schematic illustration of the system is shown in Scheme 1. Sample solutions are loaded onto the printedwax-defined testing zones of the PAD, followed by the addition of the functionalized Au-nanoprobes. After washing to remove unbound molecules, the colorimetric result can be obtained by adding the enzymatic substrate TMB to produce a visible color signal. A smartphone or a

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Figure 1. (A) A schematic of the different reagent orders to synthesize the ssDNA/HRP-AuNP and Ab-AuNP nanoprobes. (B) Colorimetric results of the different AuNP samples for HIgG detection on cellulose paper. portable CCD system can be used to capture images of the PAD test, followed by transmission and analysis to obtain quasi-quantitative results in remote settings. Optimized Functionalization of the Au-Nanoprobes Using Different Reactant Orders. Recently, studies have demonstrated that a high density of oligonucleotides can be absorbed on AuNPs using poly(A) sequences as effective anchoring blocks to the gold surface.28 The use of poly(A) as an anchoring mechanism provides several advantages. First, it eliminates the need for additional modification of DNA, so that the cost and processing requirements can be significantly reduced. Moreover, adenine is highly oxidation resistant compared with thiols for the improved longterm stability of the DNA-AuNP conjugates. ssDNA strands featuring longer poly(A) sequences provide stronger anchoring to the AuNP surfaces, but with the tradeoff of a lower density of DNA attached due to the limited surface area of the nanoparticle. Therefore, in this study we selected the oligonucleotide sequence 5’-AAAAAA-AAA-AAA-AAA-CCC-CCC-CCC-CCC-CCC-CCC-CC Biotin- 3’, which contains fifteen adenine nucleotides that can provides high surface density of ssDNA with an interstrand spacing that favors hybridization.28 For initial optimization experiments, biotinylated poly(A)/streptavidin HRP complexes and aHIgG were attached to the AuNP surfaces as a signal amplification reagent and specific detector antibody via their high binding

affinity26 and electrostatic interactions36, respectively. We characterized the functionalized Au-nanoprobes using TEM (Figure S3). Prior to surface modification, the average diameter of the synthesized AuNPs was 13 nm. After functionalization with the streptavidin-HRP bound biotinylated poly(A) ssDNA and aHIgG antibodies, the average distance between each nanoparticle increased, further confirming the successful conjugation of the ssDNA sequences on the AuNP surfaces. The order of the poly(A)/HRP and aHIgG conjugation on the AuNPs is one of the critical factors for achieving a high degree of signal amplification without sacrificing the target analyte specificity. An illustration of the procedures for synthesizing functionalized Au nanoprobes featuring different orders of surface modification is shown in Figure 1A. To test the impact of the different reactant orders when functionalizing the Au nanoprobes, we first measured different concentrations of HRP using UV−Vis absorbance spectroscopy to establish a standard curve for the HRP density on the nanoprobes (Figure S1). The absorption wavelength of the supernatant of the functionalized Au nanoprobes is centered at 650 nm, which is the absorbance of the oxidized TMB. For the Au nanoprobes made with poly(A) ssDNA sequences added first (ssDNA/HRP-AuNPs), the absorbance of the supernatant was 0.1988 and 0.3730 at 2 and 4 min after the ad-

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Figure 2. (A) Schematic of the different ratios of streptavidin-HRP to AuNPs tested. (B) Colorimetric results of the different ratios of streptavidin-HRP to biotinylated poly(A) anchored on the Au-nanoprobes for HIgG detection on cellulose paper. Table 1. Comparison of HRP concentrations of the Au-nanoprobes made using different orders of synthesis. Au-Nanoprobe Order

Concentration of HRP in Suspension [µM]

Final ratio of HRP Binding to AuNPs (AuNPs : HRP)

ssDNA/HRPAuNPs

8.79

1 : 8.5

Ab-AuNPs

9.31

1 : 3.1

dition of TMB, respectively. On the other hand, the absorbance values of the supernatant of the sample modified by the aHIgG antibody first (Ab-AuNPs) was 0.8651 and 1.2478 at these same time points (Figure S2). A summary of signal intensities from the different probe preparation processes is shown in Table 1. Our results demonstrate that the AbAuNP nanoprobes modified with the aHIgG antibody prior to the addition of the biotinylated-ssDNA results in lower poly(A) ssDNA sequences attached to the AuNPs. This can be attributed to the larger steric occupation of the antibodies on the AuNP surfaces, which leaves the poly(A) ssDNA sequences limited space on which to anchor. Therefore, modification of the AuNPs with the antibodies first hinders

poly(A) ssDNA attachment to the gold surface, resulting in a lower HRP concentration and reducing the TMB interaction. These results were also confirmed on the PAD platform using the two different orders of prepared Au nanoprobes for the HIgG immunoassays (Figure 1B). It is clear that the addition of poly(A) ssDNA sequences to modify the AuNP surfaces prior to the addition of antibodies results in a significant color change, with blue/red color ratios between 1.5–2.4. In contrast, the sample modified with the antibody first did not show any significant color change on the PAD (blue/red color values ranged from 1.0–1.2). As a result, ssDNA/HRP-AuNPs were used in all subsequent experiments, and simply referred to as Au-nanoprobes to indicate the complete functionalized ensemble (ssDNA, HRP, and antibody). Optimization of the Ratio of Streptavidin-HRP to Biotinylated-DNA Functionalized Au-Nanoprobes. The determination of the optimal molar ratio of streptavidin-HRP to the AuNPs made with a fixed amount of biotinylated poly(A) ssDNA is another critical factor in achieving efficient signal amplification in a cost-effective manner. An illustration of the optimization experiment using different ratios of streptavidin-HRP to AuNPs is shown in

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Figure 2A. Based on previous findings in the literature,28 the molar ratio of poly(A) ssDNA to the AuNPs was fixed at 100:1 in order to generate the densest configuration of erect biotinylated-DNA on the gold surfaces for streptavidin-HRP conjugation. For the optimized signal amplification, we tested three different molar ratios of streptavidinHRP to the AuNPs, including 50×, 75×, and 100×. We determined the amount of streptavidin-HRP bound to the biotinylated ssDNA-AuNPs using the mean absorbance of the supernatant solutions at 650 nm after reacting with TMB and comparing the results to the standard curve of the HRP molecules only (Figure S4). After 4 min reaction with TMB, the concentration of the three different streptavidin-HRP molar ratios (50×, 75× and 100×) was 0.56 µM, 0.99 µM, and 1.48 µM, respectively. A summary of the signal intensities of the functionalized Au-nanoprobes made using different ratios of streptavidin-HRP to biotinylatedDNA is shown in Table 2, with the results indicating the Au-nanoprobes made using a molar ratio of 100:100:1 (streptavidin-HRP/biotinylated ssDNA/AuNP) resulted in the highest HRP binding to Au (85:1). This sample resulted in the lowest percentage of lost HRP, which means the optimal ratio of streptavidin-HRP to biotinylated-ssDNAAuNP is 100:1. Meanwhile, the result also confirmed the binding affinity of biotinylated-ssDNA to the AuNPs is strong enough to resist the high speed centrifugation process used to prepare the Au-nanoprobes. Table 2. The binding amount of streptavidin-HRP on the Au-nanoprobes at different molar ratios of streptavidinHRP, biotinylated-poly(A) ssDNA, and AuNPs. Molar Ratio of the Au-Nanoprobes (HRP, ssDNA, and AuNPs)

Concentration of HRP in Suspension [µM]

Final ratio of HRP binding to Au-Nanoprobes (AuNPs and HRP)

50 : 100 : 1

9.18

1 : 4.4

75 : 100 : 1

8.97

1 : 6.5

100 : 100 : 1

8.79

1 : 8.5

In addition, we also confirmed the HRP concentration bound to the AuNPs by the color variance of the corresponding PAD test. From the HIgG colorimetric immunoassay results shown in Figure 2B, we found that the Au-nanoprobes made with an HRP molar ratio of 100 achieved a much more obvious blue color change (1.5–2.4) compared to those made using a ratio of just 50 (1.3–1.7). Based on these results, we concluded an HRP to AuNP ratio of 100 was an optimal condition for this amplification experiment.

Figure 3. Time course study for the HRP-TMB enzymatic reaction that produces color on the PAD testing zones. (A) The images and (B) data analyses of the colorimetric results of different concentrations of KLK3 at different HRPTMB enzymatic reaction times. Effect of Enzyme Reaction Time on the Signal Enhanced P-ELISA Test. For colorimetric enzymatic reactions in ELISA tests, each enzyme-substrate pair produces its own characteristic set of hues, and the appearance of the signal intensity changes with the interactive time of the substrate with the enzyme. In order to acquire optimal analytical performance without using a stop solution in order to simplify the paper-based P-ELISA testing procedure, we tested the enzyme reaction times using the optimized experimental parameters previously determined for the Aunanoprobes. These nanoprobes were used for the detection of different concentrations of KLK3 target analyte using a direct immunoassay format on the P-ELISA platform. After TMB was added for signaling, the coloration results of each testing zone were recorded for 2–8 min, individually. As shown in Figure 3, we observed the color in these zones to change from sky blue to dark blue from 2 to 6 min on the paper surface. After 6 min, the color stayed more constant. Based on our analysis of the results, we chose 4 min as the optimal TMB reaction time for colorimetric results in the signal enhanced P-ELISA system based on the maximum color change of the B/R values for the target analyte and control.

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AuNPs (100 pg/mL). Additionally, the immunoassay turnaround time can be completed in as little as 15 min, while the conventional ELISA method requires 2-4 h to complete. These results show that using AuNPs as carriers for the HRP molecules leads to better performance in comparison to the conventional P-ELISA procedure that uses HRP only as the signaling tag. Using the aforementioned optimizations of the P-ELISA test for the KLK3 antigen, our functionalized AuNPs could be used to improve the sensitivity, lower its detection limits, and also shorten the assay time, and be of particular value to medical evaluations requiring high accuracy, such as early disease diagnosis and post-surgery prognosis. We again underline that these results can be achieved simply by introducing AuNPs as multi-enzyme carriers within well-established ELISA procedures for no significant change in the experimental protocols. For this reason, the concept of Au-nanoprobes can be easily extended to other important ELISA tests on the traditional well-plate and other optical based sensing platforms.

Figure 4. Evaluation of the signal amplification effect of the signal enhanced P-ELISA test. Corresponding photographs of the paper testing zones using (A) the functionalized Au-nanoprobes and (B) the conventional ELISA method. (C) A plot of KLK3 standards measured using a conventional paper-based ELISA test and the Au-nanoprobes performed on a PAD platform. Error bars indicate standard deviations (n = 5). The Signal Amplification Effect of the Signal Enhanced P-ELISA Test. To further demonstrate the capability of our signal enhanced P-ELISA method, we used the Au-nanoprobes to detect the PSA antigen and compared its detection limit to that of a conventional paper-based ELISA test, which used the antibody-HRP interaction alone for signaling. Nine different concentrations of KLK3 antigen in buffer were measured (0.3–1000 pg/mL) using the same experimental procedures, and Figure 4 summarizes the performances of these two systems. As can be seen, when the functionalized Au nanoprobes were used, we recorded much higher colorimetric signals, demonstrating the assay’s superior sensitivity of 3 pg/mL, which was more than 1 order of sensitivity greater compared to the P-ELISA and regular ELISA assay without the use of

Figure 5. A plot of various KLK3 concentrations spiked in human serum and PBS buffer using the Au-nanoprobes on the PAD platform. Error bars indicate standard deviations (n = 5). Analysis of KLK3 Samples Spiked in Human Serum and Evaluation of Method Accuracy. In order to test the performance of the proposed Au-nanoprobes in a clinicalbased test, we spiked various concentrations of KLK3 antigen in human serum and measured the quantities using our signal enhanced P-ELISA method in order to determine the test’s accuracy in the complicated medium. As

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demonstrated in Figure 5, while the signals obtained in serum were slightly suppressed compared to those obtained in pure buffer, we could still easily differentiate the targeted analyte with a detection limit of 10 pg/mL. These results clearly show that our functionalized Au-nanoprobes can selectively identify KLK3 antigen at low concentrations even in complex serums. According to the literature, a PSA level of < 5 pg/mL is equivalent to a normal PSA value in a man after radical prostatectomy.23 The P-ELISA test with Au-nanoprobes for signal amplification could potentially be used to reassure an individual with regard to their disease status.

CONCLUSIONS In this work, we report a highly sensitive AuNP-assisted enzyme signal amplification method based on a paperbased ELISA format to detect a prostate specific antigen in human serum. AuNPs were used as carriers of the bio recognition antibody aKLK3 and HRPstreptavidin/biotinylated poly-A ssDNA sequences for the specific and sensitive analysis of KLK3, an important marker for prostate cancer prognosis. Since DNA is a natural polymer with excellent recognition ability as well as structural and functional diversity,50-53 it can serve as a versatile building block when coupled with synthetic polymers.28, 54 Moreover, given the commercial abilities in mass-producing oligonucleotides of almost any given sequence, copolymers with pure DNA sequences are readily available, featuring high purity and low cost. ssDNA sequences also offer unprecedented convenience in design flexibility and reliability in terms of quality control. Additionally, AuNPs have a large surface area, which enables a large number of biotinylated poly(A) ssDNA sequences to be anchored to the solid support, followed by streptavidinHRP molecules binding through the biotin and streptavidin interaction for enzymatic signal enhancement to detect extremely low concentrations of targeted proteins. This enzyme signal amplification strategy has proven that biotinylated poly(A) ssDNA can be incorporated with AuNPs for a simpler probe preparation process that forgoes the need for costly thiolate molecules or fluorescent-labeled ssDNA. Compared with conventional paper-based ELISA tests, the proposed sensing strategy not only results in better performance, but also shortened incubation time for the TMB color developer (15 min compared to hours for the classical ELISA procedure). The limit of detection for our signal amplification strategy is suitable as an unambiguous method for monitoring patients’ response to cancer therapies and recurrence for those who have undergone a radical prostatectomy.

Different antibodies can be introduced during the Aunanoprobe preparation process to expand the platform for multi-targeted diagnostics. In addition, different poly(A) DNA sequences can be designed and tailored for higher signal intensity with different diagnostic strategies. Moreover, other nanomaterials such as silica nanoparticles (SiNPs) can be used as the solid support based on their high modification ability and provide other functionalities. In this study, the stability of the as-prepared ssDNA/HRPAuNPs probe was dependent on attached antibodies. We have checked the activity of the functionalized Au nanoprobes can be stored at 4°C for two weeks without significantly losing their activity. For long-term storage, freezedrying can be adopted to prolong the lifetime. We expect the findings from this work can be used to build cost-effective and portable approaches for highly sensitive and specific disease diagnostics and monitoring the prognosis after medical treatment in resource limited environments.

ASSOCIATED CONTENT Supporting Information. The supporting information contains the standard curve for the HRP density on the Au-nanoprobes after addition of TMB, the absorption wavelength of the supernatant of the functionalized Au-nanoprobes with different orders of surface modification, TEM images of the functionalized Au-nanoprobes, and the absorption wavelength of the supernatant of the functionalized Au-nanoprobes with different molar ratios of streptavidin-HRP. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Chien-Fu Chen, E-mail: [email protected] *Huan-Tsung Chang, E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was supported by the Ministry of Science and Technology, Taiwan (MOST 106-2221-E-002-139-MY2 (C.F.C.) and 104-2113-M-002-008-MY3 (H.T.C.)) and the Aim for Top University Project at National Taiwan University.

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