Staining Traditional Colloidal Gold Test Strips with Pt Nanoshell

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Biological and Medical Applications of Materials and Interfaces

Staining Traditional Colloidal Gold Test Strips with Pt Nanoshell Enables Quantitative Point-of Care Testing with Simple and Portable Pressure Meter Readout Di Huang, Bingqian Lin, Yanling Song, Zhichao Guan, Jie Cheng, Zhi Zhu, and Chaoyong James Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b15562 • Publication Date (Web): 20 Dec 2018 Downloaded from http://pubs.acs.org on December 21, 2018

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ACS Applied Materials & Interfaces

Staining Traditional Colloidal Gold Test Strips with Pt Nanoshell Enables Quantitative Point-of-Care Testing with Simple and Portable Pressure Meter Readout Di Huang†, Bingqian Lin†, Yanling Song‡, Zhichao Guan§, Jie Cheng†, Zhi Zhu*,†, Chaoyong Yang*,†,‡. MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Collaborative Innovation Center of Chemistry for Energy Materials, Key Laboratory for Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China †

Institute of Molecular Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University Shanghai 200127, China. ‡

§PASSTECH

Co., Ltd. Xiamen 361005, China.

ABSTRACT: Traditional immunochromatographic test strips based on colloidal gold are effective devices for portable and low-cost point-of-care (POC) testing. Nevertheless, they still suffer from the limitation of qualitative or semi-quantitative tests via naked-eye detection. Replacement of gold with other signal entities, such as magnetic particles or fluorescent particles, requires professional instrumentation to obtain quantitative results. Pressure-based assay (PASS) with platinum nanoparticles (PtNPs) can provide quantitative results using a portable pressure meter, but also is hampered by the longterm instability of PtNPs. Consequently, we developed a Pt-staining method based on test strips to create platinum nanoshells on the surface of colloidal gold. This method not only preserves the original advantages of colloidal gold with easy synthesis and decoration, but also introduces PtNPs with excellent catalytic activity as signal labels to achieve sensitive quantitative detection. Myoglobin was tested as model target, and the LOD was 5.47 ng/mL in 20% diluted serum samples, which satisfies the requirements for clinical monitoring of acute myocardial infarction (AMI). In addition, the two most common colloidal gold strips available in the marketplace were applied to demonstrate the compatibility of Pt-staining. Taking advantage of low cost, user-friendliness, compatibility, simplicity, and stability, colloidal gold test strips with Pt-staining are expected to satisfy the need for quantitative POC testing of biomarkers especially in resourcelimited regions. KEYWORDS: colloidal gold test strips, Pt-staining, quantitative Point-of-care testing, pressure-based assay INTRODUCTION Point-of-care (POC) testing is currently one of the fastest growing areas of in vitro diagnostics (IVD). With the advantages of low cost, user-friendliness, time-saving and simplicity, POC testing plays an important role in improving the quality of medical services and fitting the healthcare needs of remote districts.1-3 To meet the specific requirements of POC testing, several practical portable analytical platforms have been introduced. For example, lateral flow immunoassay (LFIA) has shown remarkable development over the past decade, and has been applied to detection and/or diagnostics in resourcepoor or non-lab areas.4 When samples of complex matrices flow through the strip by capillary forces, the analytes first bind to the recognition molecules (antibodies usually) modified with signal labels, and then are captured by another recognition molecule. Visual detection results can be easily obtained by naked eye in a

short time with the help of signal labels, such as colloidal gold. Commercial colloidal gold strips are the most common LFIA platforms with high selectivity, low price, and easy operation for POC testing of hormones5, disease biomarkers6, toxins7, microorganisms8-9, etc. However, these popular devices are still limited to qualitative or semi-quantitative tests. But in some cases, “yes/no” results from LFIA are not sufficiently convincing for POC testing.10-11 Therefore, much effort has been devoted to improve LFIAs for quantitative detection. For example, researchers developed some image processing and analysis algorithms on a smart phone platform to provide quantitative results based on conventional LFIAs.5, 12 However, smart phone is suffering from poor endurance capacity and fancy price undoubtedly not widespread in depressed areas and remote districts desiderating for POC testing, which may limit its application prospect. At the present time, another way to

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enhance the sensitivity or the limit of detection (LOD) of LFIAs is mainly changing colloidal gold to different signal molecules and combining with other detection techniques.13-15 Several alternative labeling entities have been used with LFIA to obtain quantitative test results, such as quantum dots (QD)16-18, magnetic particles19-20, phosphorescent particles21, Raman-active materials22, and up-converting phosphor nanoparticles23. Assuredly, the detection sensitivity has been greatly improved with the help of these particles for signal amplification. For example, Wu et al.16 developed a QD-based LFIA to quantitatively detect C-reactive protein (CRP) with an LOD of 0.3 ng/mL using a fluorescence immunoassay analyzer. The method can cover two clinical ranges for both high-sensitivity and routine CRP detection. Unfortunately, all these quantitation methods rely on sophisticated instruments for signal readout, which runs counter to the needs for POC testing. Therefore, other LFIA signaling strategies are highly desired for rapid detection of targets with high sensitivity without the use of professional equipment. Gas generation-induced Pressure-based assay (PASS) is an attractive method for POC testing, which integrates target recognition biosensors with catalyzed gas-generating reactions.24-29 The pressure can be measured easily by a portable pressure meter like the ones frequently used in our daily lives.30-31 The decomposition of H2O2 to O2 is one of the most wide-applied gas generating reaction with environment-friendliness and nontoxicity of the reaction and its products, which is suitable for POC testing. Compared with catalase, one of the most efficient enzymes found in nature, the PtNPs were still shown to be highly efficient catalyzer for such reaction that receive widespread attention in bioanalysis.31-35 Lin et al.11 applied PtNPs for signal transduction and amplification in LFIA. After PtNPs-catalyzed gas generation in a gas-tight container, quantitative results were achieved by direct measurement of pressure using a portable pressure meter. However, non-specific adsorption of PtNPs on porous surfaces results in high background for irreproducible results. In particular, PtNPs are highly prone to aggregation and poisoning during long-term storage. And sometimes the catalytic activity of PtNPs decreases significantly after modification with recognition molecules, such as antibodies and aptamers. In order to overcome the problems associated with PASS using PtNPs, Li et al.36 developed a Pt-staining method. Gold nanopaticles (AuNPs) are stained with silver and platinum bimetallic nanoshells (Au@AgPtNPs) to provide AuNPs with enhanced properties. The freshly made Au@AgPtNPs are liberated from the instability with time and exhibit increased catalytic ability because of the electronic synergetic effects between the metallic layers. In this way, they transformed AuNPs-labeled enzymelinked immunosorbent assays (ELISA) into pressurebased methods. Inspired by the work of Li et al., we have introduced Ptstaining into colloidal gold test strips to transform visible

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qualitative or semi-quantitative detection into quantitative results. We used a standard colloidal gold test strip to analyze the targets. And then the test line which capture the targets with colloidal gold labelled antibodies was cut off. After that, Pt-staining was operated on the surface of colloidal gold on test line generating Au@AgPtNPs. Finally, Au@AgPtNPs were transferred to a tight container containing H2O2 to catalyze the gas production reaction. The pressure increase is directly related to the target concentration. In this manner, the advantages of colloidal gold, including easy synthesis and decoration, are maintained. At the same time, the unique catalytic ability of PtNPs is achieved by a convenient Pt-staining process instead of using PtNPs as signal molecules, thereby avoiding the analytical defects mentioned above. Note that the portability and simplicity of LFIA test strips are not sacrificed. Without the need for sophisticated instrumentation, quantitative results are obtained by a simple and portable pressure meter, which satisfies the need of POC testing. Last but not least, our method is expected to have excellent compatibility with any commercial colloidal gold test strips for quantitative detection results after Pt-staining. In this work, we chose myoglobin (Myo) as a model analyte, an early biomarker for acute myocardial infarction (AMI), to confirm the feasibility of Pt-staining on colloidal gold test strips for pressure-based quantitative detection. The level of Myo in a patient’s blood increases more dramatically than the concentrations of the other two AMI biomarkers, cardiac troponin I (cTnI) and creatine kinase MB isoenzyme (CKMB). The excessive level of Myo remains steady for 24 h. Consequently, it is significant and highly desired that quantitative POC testing for Myo be achieved within this time frame, before suspected AMI patients suffer irreparable myocardial cell damage.37-38 In order to verify the compatibility of the method, we purchased the two most common colloidal gold strips available commercially, “pregnancy test strips” and “ovulation test strips”, and applied Pt-staining for quantitative detection of human chorionic gonadotropin (HCG) and luteinizing hormone (LH), respectively.

EXPERIMENTAL SECTION Reagents and materials. Monoclonal capture antibody and detection antibody for Myo, redissolution buffer of colloidal gold, pressure generating and measuring devices were obtained from PASSTECH Co. Ltd. (Xiamen, China). Myo and LH were obtained from R&D Systems Inc. (Minneapolis, MN, USA). HCG was purchased from Sino Biological Inc. (Beijing, China). Bovine serum albumin (BSA) was purchased from Shanghai Seebio Biotech Inc (Shanghai, China). Tween-20 was purchased from SigmaAldrich (St. Louis, MO, USA). PBS buffer (1x) was purchased from Boster-bio (Wuhan, China). Other reagents were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).


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For making test strips, NCM were purchased from Shanghai Kinbio Tech. Co. Ltd. (Shanghai, China), and PVC backing, polyester membrane, glass fiber membrane, and absorbent paper were all obtained from Shanghai Jiening Biological Technology Co. Ltd. (Shanghai, China). Commercial HCG-responsive test strips were purchased from Runbio. Biotech Co. Ltd. and Beijing Jinhuake Biotech Co. Ltd., while LH test strips from Wondfo Biotech Co. Ltd.. Gas-generating equipment and pressure meter. As shown in Fig. S1, the gas-generating equipment consisted of gastight container, eight-well ELISA plate and rubber strip. The container was compatible with commercial eight-well ELISA plate, and eight samples can be held and analyzed synchronously. In order to keep the gas-generating wells with constant volume, a flat of rubber strip was introduced as a sealing plug. As the rubber strip was slightly protuberant over the brim when covered the eight-well ELISA plate, tightness was guaranteed after closing the shell of the container. After gas-generating reaction, a handheld pressure meter was used to measure the gas pressure in wells. The pressure meter was assembled with a LED screen, a lithium battery with endurance capacity, and a sensitive atmospheric pressure sensor connected with a 0.7 mm needle to pierce the rubber strip and measure the atmosphere in wells (Fig. S1B). This portable meter was able to measure the pressure ranging from 0 to 3000 kPa with an outstanding accuracy (0.01 kPa), satisfying the need for quantitative POC testing. Colloidal gold synthesis and modification. Colloidal gold was synthesized according to a previous literature procedure.39 All glassware used in synthesis were cleaned with fresh aqua regia and then thoroughly rinsed with H2O. HAuCl4 (2.5 mL, 1 wt%) and H2O (93.5 mL) were poured into a round flask with stirring, followed by heating and refluxing. When the solution was boiling, sodium citrate (4 mL, 1 wt%) was added immediately. After boiling for 10 min, colloidal gold particles with a size of about 28 nm were acquired. After cooling to room temperature, the colloidal gold solution was stored at 4 °C for subsequent use. Detection antibody (DA) for Myo was modified on the surface of colloidal gold by physical adsorption. Colloidal gold (1 mL) and K2CO3 (5 μL 0.2 M) were placed in a tube, followed by mixing and incubation for 5 min. Subsequently, DA for Myo (100 μL, 10 μg/mL) was added in small portions, and the mixture was incubated for 10 min after blending. In order to block the remaining sites on colloidal gold, BSA solution (10 μL, 10 wt%) was added and incubated overnight at 4 °C. After centrifugation to remove the supernatant, modified colloidal gold was resuspended in 0.6 mL redissolution buffer and stored at 4 °C for further use. Working principle of Pt-staining on the test strips. The working principle of Pt-staining on the test strips is shown in Fig. 1. Traditional colloidal gold test strips are

used to recognize and capture target to achieve qualitative or semi-quantitative results. Sample is added to the sample pad and moves to the right by capillary forces. When target is present, it binds with colloidal gold-DA on the conjugate pad and continues to flow by capillary forces until being captured by capture antibody on the test line (T). The unbound colloidal gold-DA is captured by the secondary antibody on control line (C). Different shades of red color on test lines are related to the amount of captured colloidal gold-DA, in turn reflecting the different concentrations of target in the samples. Preparation of Myo-responsive test strips. Test strips are mainly composed of 5 parts: sample pad (13 mm), conjugate pad (6 mm), nitrocellulose membrane (NCM) (25 mm), absorption pad (22 mm), and polyvinyl chloride (PVC) sheet. The sample pad was made of a glass fiber membrane, which was used to separate the impurities, adjust the penetration rate, and promote uniform distribution of samples. The conjugate pad was made of a polyester membrane to immobilize colloidal gold-DA of Myo and reduce non-specific adsorption. For improving the stability of bonding, the conjugate pad was pretreated with 6% sucrose solution. And then colloidal gold-DA was sprayed on the polyester membrane, followed by drying in a vacuum furnace at 30 °C for 4 h. NCM was used to immobilize the capture antibody and the secondary antibody of Myo. After being diluted in PBS buffer respectively, the antibodies were dispensed onto the NCM in the amount of 1 μL/cm and formed two reaction lines, named test line and control line. The ready-made NCM was dried overnight at 37 °C at constant humidity of 25– 30% RH. The absorption pad provided the ability to control the flow rate and promote capillary function. Finally, the sample pad, conjugate pad, NCM, and absorption pad were pasted on a PVC sheet overlapping in sequence. After cutting into thin strips with the width of 2 mm by a cutting machine, the basic structure of test strips was produced. Optimization of Pt-Staining Condition. The Pt staining was performed by first reacting the test line containing colloidal gold with Ag precursor (AgNO3) and reducing agent (hydroquinone) to form an Ag shell on the surface (Au@AgNPs). And then, Pt precursor (H2PtCl6) and reducing agent (ascorbic acid) was used to coat Au@AgNPs with a Pt nanoshell (Au@AgPtNPs). If without target, the colloidal gold-DA would accumulate on the control line, which was used to optimize the Ptstaining condition for best signal to background ratio (S/N). Test strips without colloidal gold-DA were regarded as control groups. First, by holding the end of absorption pad, the test strips were dipped vertically into 30 μL reaction buffer (0.9 wt% NaCl, 0.1 wt% Tween-20) without Myo for 5 s. When the liquids moved to the NCM, the test strips were removed and placed flat on a non-adsorbing surface for 10 min. The



control lines were cut off and added to tubes containing AgNO3 (50 µL, 0-3.2 mM) and hydroquinone (50 µL, 0-40 mM). After incubating at 37 °C for 20 min, the cut piece in each tube was washed with 100 µL H2O twice. Then, H2PtCl6 (50 µL, 0-4 mM) and ascorbic acid (50 µL, 0-40 mM) were added to each tube and incubated at 37 °C for 20 min. After washing, the cut pieces were transferred to an airtight container and reacted with H2O2 (100 μL, 30

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wt%) for 30 min at room temperature. Finally, the pressure was measured with a pressure meter. The pressure signals produced by control lines with colloidal gold, and the pressure background produced by bare control lines were shown in Fig. S3-4. Considering the S/N, the optimum concentrations for AgNO3, hydroquinone, H2PtCl6 and ascorbic acid were 2.5 mM, 20 mM, 1.0 mM and 20 mM, respectively.

Figure 1. Working principle of colloidal gold test strips with Pt-staining for quantitative POC testing based on pressure meter readout.

RESULTS AND DISCUSSION Working process of Pt-staining on the test strips. The working process of Pt-staining on the test strips is shown in Fig. 2A and Fig. S2. Pt-staining is performed right away to obtain quantitative results. First, the test line is cut off from the test strip, and colloidal gold on the test line reacts with Ag precursor (AgNO3) and reducing agent (hydroquinone) to form an Ag shell on the surface (Au@AgNPs). Afterwards, with the help of the Ag shell, Pt precursor (H2PtCl6) and reducing agent (ascorbic acid) can coat Au@AgNPs with a Pt nanoshell (Au@AgPtNPs).

After catalytic gas generation of decomposing H2O2 by Au@AgPtNPs in a tight container, the pressure value can be easily obtained by a portable pressure meter. The pressure increases correlates directly with the content of Au@AgPtNPs on the test line, and thus it is also proportional to the concentration of target. In this way, quantitative detection can be achieved using colloidal gold test strips and a handy Pt-staining post-treatment, without any professional instrumentation. Because Ptstaining is performed immediately, it avoids the PtNPs aggregation or poisoning during storage. Therefore, there is no decrease in the catalytic activity.

Figure 2. A). The whole process of Pt-staining on the colloidal gold test strip for quantitative POC testing. B). Linear response of ∆P vs Myo concentration (0, 6.67, 16.67, 33.33, 66.67, 100, 150 ng/mL) in buffer with standard deviations obtained from three measurements (R2=0.979). Feasibility of Pt-staining on the surface of colloidal gold. In order to investigate the feasibility of Pt-staining on the surface of DA-modified colloidal gold, we carried out

these processes in solution phase and characterized the structures of colloidal gold-DA and products at each step (Au@AgNPs, Au@AgPtNPs) by TEM and STEM. As


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shown in Fig. S5 and S6, the size of colloidal gold-DA was about 28 nm, and the size of Au@AgNPs was increased visibly after coating with Ag shells. Finally, after coating with rough and loose Pt nanoshells, Au@AgPtNPs were created with sizes about 52 nm. Furthermore, with the help of energy-dispersive X-ray spectroscopy (EDS), Fig. S7 showed elemental position of the three types of nanoparticles, which further confirmed the feasibility of Pt-staining on the surface of colloidal gold. Feasibility of Pt-staining on the test strips. To confirm the feasibility of Pt-staining on the test strips, seven samples in 30 μL reaction buffer with different concentrations of Myo were tested by Myo-responsive colloidal gold test strips. As shown in Fig. 2A, by holding the absorbent pads, firstly the test strips were dipped vertically into samples. When the colloidal gold solution moved to the NCM, test strips were removed and placed horizontally for 5 min. Visual detection of different shades of red color on the test lines reflected the different concentrations of Myo. And then, test lines were cut off and added to tubes for Pt-staining. Finally, the catalytic effect of each Ptstained nanoparticle on the test line was evaluated by measuring the pressure change with a pressure meter. As shown in Fig. 2B, the pressure changes (∆P) compared with a blank sample (0 ng/mL Myo) exhibited a linear increase with increasing concentration of Myo ranging from 6.67 to 150 ng/mL. The standard curve showed an acceptable fit (R2=0.979) with a detection limit of 3.82 ng/mL by the definition of 3σ/slope, where σ is the standard deviation of blank samples.

Figure 3. The specificity of Pt-stained test strips for Myo detection with three repeated measurement. 50 ng/mL for Myo, 500 ng/mL for other proteins. The specificity of Pt-stained test strips. Since complex matrices in blood samples may interfere with Myo

detection, it is important that test strips retain their high selectivity for Myo after Pt-staining. To demonstrate the specificity of our method, the other two biomarkers for AMI (CKMB, cTnI), and some common proteins in human blood, including C-reactive protein (CRP), alkaline phosphatase (ALP), transferrin (TRF) and human serum albumin (HSA), were chosen as negative controls. As shown in Fig. 3, only Myo could effectively produce an obvious pressure increase, even when the concentrations of control proteins were 10 times that of Myo. These results clearly demonstrated the specificity of the Ptstained test strips for Myo detection. The performance of Pt-stained test strips in 20% human serum. Myo in human serum samples was tested to further verify the clinical application of Pt-stained test strips. A series of concentrations of Myo from 0-150 ng/mL were spiked into 20% human serum from a normal volunteer having a low background concentration of Myo (28.7 ng/mL in undiluted serum) measured by chemiluminescent microparticle immunoassay (CMIA). The experimental procedures were the same as described above. As shown in Fig. 4A, ∆P represents the pressure change relative to the blank sample (no Myo in the presence of 20% normal serum). A linear relation between the pressure changes and the concentrations of Myo spiked in the human serum samples was obtained, indicating good performance of Pt-stained test strips in real sample detection. By calculation, the LOD for Myo in human serum was 5.47 ng/ mL, and the standard curve showed a fit (R2=0.981), similar to that in buffer solution. The dynamic range was from 5.74 to 150 ng/mL in 20% diluted human serum, indicating that the actual detection range of clinical samples is from 28.7 to 750 ng/mL. Since the clinical cut-off value for Myo is 70-200 ng/mL, Ptstained test strips clearly satisfy the clinical quantitative detection requirement of AMI.40 Note that total volume of sample is 30 μL, which means that only 6 μL serum is required without any pretreatment in our method, while the serum volume used in CMIA is 80 μL.11 Detection of Myo in clinical samples using Pt-staining test strips. After demonstrating the feasibility of detecting Myo in diluted human serum using Pt-stained test strips, we further compared the developed method with CMIA, the “gold standard” of practical detection of Myo in medical institutions. Ten real serum samples from First Affiliated Hospital of Xiamen University, including 5 negative samples and 5 positive samples, with 28.7-711.4 ng/mL of Myo were analyzed by both CMIA and Pt-stained test strips.



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Figure 4. A). Linear response of ∆P vs spiked Myo concentration (0, 10, 20, 50, 80, 100 ng/mL) in 20% normal human serum with standard deviations obtained from three measurements (R2=0.981). B) Correlation analysis between the results of Pt-stained test strips and standard clinical method CMIA for Myo detection in 10 real serum samples. (R2=0.987). C) Bland-Altman analysis for the agreement between Pt-stained test strips and clinical results of 10 real serum samples. The 95% confidence interval of the mean is shown on the graph. Detection results of Myo in real serum samples are shown in Fig. 4B. The linear relation between the results by CMIA and Pt-stained test strips indicate that the two methods are strongly correlated with an R2 value of 0.987. In addition, the Bland–Altman plots (Fig. 4C) were analyzed to verify the agreement between the two methods, where each scatter point indicates the difference from the average result of the two methods. In Fig. 4C, the blue horizontal line represents the mean difference, and the two dotted lines showed limits of agreement at a 95% confidence level defined as the mean difference ± 1.96 standard deviation (SD), if the difference obeys a Gaussian distribution. Pt-stained test strips showed a bias of - 7.5 ng/mL at the limit of agreement from 49.1 to - 64.1 ng/mL for Myo detection compared to the CMIA. According to these results, Pt-stained test strips can reliably detect Myo in real serum samples, indicating the possibility of early diagnosis of acute myocardial infarction.

sensitivity of visual semi-quantitative detection was improved after Pt-staining (Fig. 6A). Catalytic gas generation provided acceptable quantitative results, as shown in Fig. 6B. ∆P showed a corresponding linear increase with increasing concentrations of LH with a correlation coefficient (R2) of 0.988, and the calculated LOD was 2.94 mIU/mL. We also performed the detection using three different batches of HCG test strips from the same company, demonstrating the acceptable reproducibility of Pt-staining for commercial test strips (Fig. S8). Consequently, Pt-staining is compatible with commercial colloidal gold test strips for quantitative detection, thereby exhibiting the outstanding potential of Pt-staining for POC testing.

The compatibility of Pt-staining with commercial test strips. There are numerous test strips on the market to provide qualitative detection for a variety of targets. If Pt-staining is also applicable to commercial colloidal gold test strips for quantitative detection, it will greatly expand the application of these traditional detection devices in POC testing. In order to verify the compatibility of our method, commercial “pregnancy test strips” and “ovulation test strips” were combined with Pt-staining for quantitative detection of HCG and LH, respectively. First, 50 μL reaction buffer with different concentrations of HCG from 0-1213.5 mIU/mL were tested by commercial “pregnancy test strips”. After Pt-staining, visual semiquantitative results (Fig. 5A) demonstrated the improved sensitivity. Quantitative results were easily achieved by catalytic gas generation as mentioned previously. As shown in Fig. 5B, ∆P increased linearly with the increasing concentrations of HCG with a correlation coefficient (R2) of 0.974, and the calculated LOD was 3.50 mIU/mL. Analogously, 50 μL reaction buffer with different concentrations of LH from 0-200 mIU/mL were tested by commercial “ovulation test strips”. Similarly, the

Figure 5. The compatibility of Pt-staining with commercial test strips. A). Visual detection results of HCG by colloidal gold test strips and after each step of Pt-staining (Au@AgNPs and Au@AgPtNPs). B). Linear response of ∆P vs HCG concentration in buffer with standard deviations obtained from three measurements (R2=0.974).


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Comparison with image processing quantitative methods. Some researches combined colloidal test strips with image processing, which was a conventional quantification method based on visual detection results, and achieved outstanding detection sensitivity. We have compared the performance of Pt-staining with the image processing result (ImageJ). Taking LH test strips as an example, the gray values of test lines with the present of different concentrations of LH were analyzed. As shown in Fig. 6C, gray values showed a corresponding linear with

concentrations of LH (R2=0.989), and LOD was 19.12 mIU/mL, which is six times higher than that of Ptstaining. Therefore, in terms of detection sensitivity, Ptstaining was obviously better than gray value analysis, which indicated the application potential of the Ptstaining. Besides, as shown in Table. S1, Pt-staining was compared with other methods with smartphones.5, 12 Ptstaining have comparable sensitivity with other reports but reduced cost that can better satisfy the need for POC testing.

Figure 6. A). Visual detection results of LH by colloidal gold test strips and after each step of Pt-staining (Au@AgNPs and Au@AgPtNPs). B). Linear response of ∆P vs LH concentration in buffer with standard deviations obtained from three measurements (R2=0.988). C). Linear response of gray value vs LH concentration in buffer with standard deviations obtained from three measurements (R2=0.989). Supporting Information

CONCLUSIONS In summary, we have integrated traditional colloidal gold test strips with Pt-staining to achieve quantitative detection of specific targets with high selectivity and sensitivity. While maintaining the advantages of colloidal gold, including easy synthesis and decoration, PtNPs with unique catalytic ability are attached by a convenient Ptstaining process. Because Au@AgPtNPs are created in real time, aggregation and poisoning issues are avoided. Therefore, quantitative results based on catalytic gas generation by PtNPs are achieved, while the portability and simplicity of LFIA test strips are preserved. We chose Myo as a model analyte and made Myo-responsive colloidal gold test strips. After Pt-staining and catalytic gas generation, Myo in 20% diluted serum samples was readily detected by a commercial hand-held pressure meter with an LOD of 5.47 ng/mL, thereby satisfying the exacting standard for AMI POC testing in risk assessment. Furthermore, this novel Pt-staining post-treatment provides excellent compatibility. Commercial “pregnancy test strips” and “ovulation test strips” were also adapted for sensitive quantitative detection results. The LODs for HCG and LH were 3.50 mIU/mL and 2.49 mIU/mL, respectively. The sensitivities were significantly improved compared with visual detection of the aforementioned colloidal gold test strips. Therefore, with the advantages of low cost, user-friendliness, compatibility, simplicity and stability, colloidal gold test strips with Pt-staining are expected to satisfy the need for quantitative POC testing of biomarkers, especially in resource-limited regions.

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Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Phone: +86 592-218-7601. Fax: +86 592-218-9959. E-mail: [email protected]. *Phone: +86 592-218-7601. Fax: +86 592-218-9959. E-mail: [email protected].

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank the National Natural Science Foundation of China (21435004, 21735004, 21775128, 21521004, 21705024, 91313302, 21325522, 21422506), Program for Changjiang Scholars and Innovative Research Teams in University (IRT13036), and the Class General Financial Grant from the China Postdoctoral Science Foundation (2017M622057) for financial support.

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