Peptide-mediated controllable crosslinking of gold nanoparticles for

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Peptide-mediated controllable crosslinking of gold nanoparticles for immunoassays with tunable detection range Bei Ran, Wenshu Zheng, Mingling Dong, Yunlei Xianyu, Yiping Chen, Jing Wu, Zhiyong Qian, and Xingyu Jiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01760 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 7, 2018

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Analytical Chemistry

Peptide-mediated controllable crosslinking of gold nanoparticles for immunoassays with tunable detection range Bei Ran †,‡,#, Wenshu Zheng ‡,#, Mingling Dong †,‡, Yunlei Xianyu ‡, Yiping Chen *,‡, Jing Wu ‡, Zhiyong Qian *,†, and Xingyu Jiang *,‡,§ †State

Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center. Chengdu, Sichuan, 610041, P. R. China ‡Beijing Engineering Research Center for BioNanotechnology and CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for NanoScience and Technology, No. 11 Zhongguancun Beiyitiao, Beijing 100190, P. R. China §The University of Chinese Academy of Sciences, 19 A Yuquan Road, Shijingshan District, Beijing, 100049, P. R. China Corresponding Authors E. mail: [email protected] (YP Chen), phone number: (86)10 82545631; E. mail: [email protected] (ZY Qian), phone number: (86)28 85501986; E. mail: [email protected] (XY Jiang), phone number: (86)10 82545558. #These authors contribute to this work equally. ABSTRACT: The colorimetric immunoassay based on gold nanoparticles (AuNPs) can hardly enable simultaneous detection of multiple biomarkers in vastly different concentrations (e.g. pg/mL-µg/mL) due to its narrow dynamic range. In this work, we demonstrate an immunoassay with tunable detection range by using peptide-mediated controlled aggregation of surface modification-free AuNPs. Alkaline phosphatase (ALP) removes the phosphate group of the peptide to yield a positively charged product, which triggers the aggregation of negatively charged AuNPs and the color change of the AuNPs solution from red to blue with naked-eye readout. We design and screen 20 kinds of phosphorylated peptides to obtain a broad and controllable detection range for ALP sensing, and apply them for detecting multiple inflammatory biomarkers in clinical samples. Our assay realizes straightforward, multiplexed and simultaneous detection of multiple clinical biomarkers with tunable detection range (from pg/mL to µg/mL) in the same run, and holds great potential for chemical/biochemical analysis.

Straightforward and simultaneous detection of the different but relevant analytes without bulk equipment are of great significance in the fields of chemical analysis, clinical diagnosis, environmental monitoring, food safety and so on.1-5 For instance, simultaneous detection of three common infectious biomarkers with broadly ranged concentration (from pg/ml-µg/mL) including procalcitonin (PCT), interleukin-6 (IL-6) and C-reactive protein (CRP) can enable much more precise diagnosis and efficiently avoid the antibiotics abuse.6 Although many efforts have been made on the development of biochemical assay, the major challenge in the current methods is the broadly ranged concentrations of the different analytes from µg/mL level to pg/mL level in complicated samples.7-13 The assay that enables the simultaneous detection of different analytes requires not only high sensitivity but also the ability to tune the detection range with convenient operation. Ultra-sensitive assays with board detection range can enable multiple detection, but they mostly rely on skilled operator and bulk equipment.14-16 Owing to their unique optical properties and facile preparation, gold nanoparticles (AuNPs) have been widely employed to develop analytical platforms.17-20 The colorimetric assays based on the AuNPs

are popular because they can enable naked eyes detection and are free of bulk instruments,21-29 and many analytical platforms have been developed by combining enzyme-linked immunosorbent assay (ELISA) with AuNPs-based colorimetric assays.30-31 However, most AuNPs-based ELISA platforms require additional employment of enzymes that are not typically accessible in antibody-conjugated forms where additional labeling for enzyme is needed, such as glucose oxidase, amylase and sucrose,32-33 which can hardly accommodate conventional ELISA. To meet this challenge, we have developed AuNPs-based ELISA using alkaline phosphatase (ALP) and horseradish peroxidase for detection of disease-related biomarkers.9,24 But a number of defects still exist including the complicated surface modification of AuNPs, the relatively tedious procedures of enzymatic cascade reactions. Besides, the narrow detection range for these assays is still one major bottleneck that limits these assays in quantitatively detecting different biomarkers with board ranges of concentrations (e.g. pg/mL-µg/mL).34-35 Development of the AuNPs-assays with tunable detection range and convenient operation for simultaneous detection of different but relevant analytes is still challenging. To

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overcome this problem, common assays typically pay attention to sample pretreatment rather than improve the response range of AuNPs for signal readout. However, these strategies unavoidably result in complex operation. Sample preparation can make the procedure more cumbersome and resulting in amount of problems. As mentioned above, improve the dynamic range for detection shows irreplaceable advances than sample pre-treatment especially for the rare samples. In this work, via the design and screen of a series of peptides, our peptide-mediated AuNPs-immunoassay can overcome these problems by broadening the response range of AuNPs with a straightforward way. Such assay enables a broad dynamic range from pg/mL to µg/mL, and the AuNPs no need any surface modification, which shows great potential in developing automated and convenient detection for multiple targets. In addition, compared to recent reported AuNPs-based assays with board detection range,36-37 our assay relies no bulk equipment or additional surface modifications. The interaction between the enzyme and peptide has drawn increasing attention in biochemical analysis. Different peptides not only response differently to the same enzyme, but also show distinct influence on the dispersed state of AuNPs to distinguish the color of AuNPs solution.38-40 Alkaline phosphatase (ALP), as a widely used labeling enzyme in immunoassays, can efficiently remove the negatively charged phosphate group from the phosphorylated peptides and alter the electrostatic property of these peptides to crosslink the dispersed negatively charged modified AuNPs.41 The state change of AuNPs (from dispersed to aggregated state) produces the color change and can be employed as signal readout. These properties inspire us to utilize enzyme and structure-controlled peptide to regulate the AuNPs aggregation, and provide a feasible way to improve the sensitivity and tune the detection range of the same assay. We employ ALP, AuNPs, and a series of phosphorylated short peptides that possess several kinds of amino acids, including cysteine (Cys), tyrosine (Tyr), serine (Ser), lysine (Lys) and arginine (Arg) to conduct the peptide-ALP-AuNPs immunoassay (PAAI) with tunable detection range. We design a series of peptides and study their different response to ALP, and choose three types of peptides to trigger the AuNPs aggregation after incubating with the same [ALP] (Fig. 1A). We justify the practicability of PAAI by using the most sensitive peptide, moderately sensitive and most insensitive peptide to broaden the detection range of assay, which enable simultaneous detection of three inflammatory biomarkers (IL-6, PCT, CRP) in clinical serum samples with tunable detection range from pg/mL to µg/mL. Furthermore, AuNPs require no surface modification in our assay, which also makes this assay easy-to-operate with good stability.

PCT are from Abcam (UK). Interleukin-6 (IL-6) (1 mg/mL), the capture antibody (5 mg/mL) and detection antibody (5 mg/mL) for IL-6 are from invitrogen (USA). Bovine serum albumin (BSA), human IgG (5 µg/mL), streptavidin (SA), biotin, alkaline phosphatase (ALP) and p-Nitrophenyl phosphate (PNPP) are from Sigma Aldrich (USA). The trisodium citrate is from Merck (Germany). All the phosphorylated peptides (purity>98%) are from TOP biological Technology Co. (Shanghai,China). PBS: 0.01 M PBS, pH 7.4; PBST: 0.01 M PBS with 0.5% Tween-20; Carbonate buffer: 0.2 M sodium carbonate/bicarbonate, pH 9.6; Blocking buffer: 3% BSA. All reagents are of analytical grade for all experiments and used without further purification.

Figure 1. The principle of peptide-ALP-AuNPs immunoassay (PAAI) for simultaneous detection of the inflammatory markers (IL-6, PCT and CRP). (A) The scheme of AuNPs-based signal read-out. Negatively charged phosphotyrosine or phosphoserine in the short peptides act as a molecular switch for the AuNPs aggregation. When ALP removes the negatively charged phosphate group from the peptide, AuNPs aggregation is triggered after addition of the resulting dephosphorylated peptide. (B) The scheme of PAAI based on three kinds of peptides for simultaneous detection of IL-6, PCT and CRP. The screening of peptides. We incubate 20 kinds of peptide solution (60 µM) with different amounts of ALP (0, 2.34, 4.68, 9.37, 18.7, 37.5, 75, and 150 U/L) in HEPES buffer solution. After 30 min of enzyme treatment, we add the resulting mixture (200 µL) to the solution of AuNPs (100 µL, 30 nm) to induce the aggregation of AuNPs. The procedure of PAAI for CRP detection. We modify the 96-well plates with capture antibody (100 µL) diluted 1:4000 (5 mg/mL) in a carbonate buffer (0.2 M sodium carbonate/bicarbonate, pH 9.6) at 4 °C overnight. After three times washing with PBST (0.01 M PBS, with 0.5% Tween-20), we block these plates with blocking buffer (3 % wt BSA in PBS) for 2 h at 37 °C. Subsequently, we wash the plates three times and add diluted solution of CRP to each well. After 1 h incubation and additional three round of

EXPERIMENTAL SECTION Materials and Equipment. Chloroauric acid (HAuCl43H2O) is from Macklin (China). C-reactive protein (CRP) (5 mg/mL), the capture antibody (5 mg/mL) and detection antibody (5 mg/mL) for CRP are from Calbiotech (USA). Procalcitonin (PCT) (2 mg/mL), the capture antibody (5 mg/mL) and detection antibody (5 mg/mL) for

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washing, we add the solution of diluted (1:4000) biotin-labeled detection antibody (100 µL) and further incubate for 1 h at 37 °C. After three times washing, we add the SA-biotin-ALP conjugate (100 µL) diluted 1:2000 for 1 h at 37 °C. After another three rounds of washing, peptide 1 (CYR) diluted 1:500 (100 µL) in water is added for 1 h at 37 °C. We add the solution of AuNPs (100 µL) to each well at room temperature. After 15 min, we collect the spectra of the solutions by UV-vis spectrometry.

spectrometry. Among these peptides, the four of them could cause obvious aggregation of AuNPs after treatment with ALP (Fig. 3), including: Cys(SH)-Ser(PO32-)-Lys(NH2)-Ser(PO32-)-Lys(NH2) (CSKSK), Cys(SH)-Tyr(PO32-)-Arg(N3H4)(CYR), Cys(SH)-Arg(N3H4)-Tyr(PO32-)-Arg(N3H4)(CRYR), Cys(SH)-Tyr(PO32-)-Lys(NH2)(CYK). The polypeptide structure can influence the degree of AuNPs aggregation in many aspects: i) The amount and position of the phosphate group, as well as the length of the peptide can determine the efficiency of ALP hydrolysis via the steric effects; ii) after hydrolysis, different degree of charging as a result of arginine or lysine will affect the degree of aggregation of AuNPs by electrostatic effects; iii) the conformation of the peptide, as building blocks crosslink also affect the degree of aggregation. Synergistic effects of these factors thus ultimately affect the spectrum of AuNPs and detection range,44-46 which open a chance to broaden the detection range of assay to realize simultaneous detection of multiplexed biomarkers in different concentrations.

RESULTS AND DISCUSSION The Design of the Short Peptides. We design 20 kinds of short phosphate group-containing peptides that contain several amino acids, including the N-terminal amino acid (Cys), whose sulfhydryl group can conjugate to AuNPs; the negatively charged phosphate group-containing amino acid (Tyr or Ser), whose hydroxy group can be modified with phosphate group; and the positively charged alkaline group-contained amino acid (Arg or Lys) (Fig. 2A and B). Since the AuNPs we used there are prepared via citrate reduction, the AuNPs are negatively charged and well dispersed due to the surface modification of citric acid. The presence of the negatively charged phosphate group next to alkaline group on the arginine (Arg) or lysine (Lys) inhibits the binding between AuNPs via Au-N bond, AuNPs would remain dispersed when mixed with the initial phosphate peptide. After adding ALP into solution, it can efficiently remove the negatively charged phosphate group from these peptides by ALP-catalyzed enzymatic dephosphorylation, and the whole peptides will become positively charged, which induces the aggregation of AuNPs by the crosslinking between the dispersed negatively charged citric acid-AuNPs.42-43 The state change of AuNPs results in a visual color change from red to blue to enable naked eyes-read out and quantitation by UV-vis spectroscopy.

Figure 3. The structure of the four kinds of different peptides that used for ALP sensing. (A) The peptide 0: Cys-Tyr-Lys (CYK). (B) The peptide 1: Cys-Ser-Lys-Ser-Lys (CSKSK). (C) The peptide 2: Cys-Tyr-Arg (CYR). (D) The peptide 3: Cys-Arg-Tyr-Arg (CRYR). We demonstrate that four peptides have different responses for ALP sensing (Fig.3), and ALP treatment successfully turns all these four kinds of negatively charged peptides into the positively charged peptides (Table S1). ALP-treated CSKSK can induce gradual shift of the absorbance peak of AuNPs from 530 nm to 600 nm with the increasing concentrations of ALP from 0 to 150 U/L, and we use this ratio between the absorbance at 600 nm and that at 530 nm (A600/A530) to quantitatively represent the degrees of aggregation for AuNPs. We can obtain a linear relationship between the A600/A530 ratio and the log concentration of ALP between 9 and 75 U/L (Fig. 4A). Similarly, we can obtain linear relationship between the A600/A530 ratio and the log concentration of ALP from 2 to 37.5 U/L (Fig. 4B), 0.5 to 18 U/L (Fig. 4C) for CYR and CRYR groups respectively. When we use ALP-treated CYK to aggregate AuNPs, the absorbance peak of AuNPs gradually shifts from 530 nm to 600 nm with the increasing concentrations of ALP from 0 to 150 U/L. However, we cannot obtain the linear relationship between the A600/A530 ratio and the ALP concentration (Fig. 4D). Based on these

Figure 2. The chemical structures of the amino acids and the peptides. (A) The five kinds of amino acids (Cys, Tyr, Ser, Arg, Lys). (B) The structures of 20 kinds of short peptides. The screening of peptides. We separately incubate 20 kinds of peptide solutions with different [ALP] to study their ability to result in the aggregation of AuNPs for developing an immunoassay with tunable detection range. After enzyme treatment for 30 min, we add the resulting peptide solutions to the AuNPs suspension to induce aggregation. We investigate the response of peptides to [ALP] by means of the color of AuNPs and UV-vis

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peptide-ALP-AuNPs (PAA) reaction systems and their different analytical performances for ALP sensing, we develop PAA immunoassay (PAAI) for CRP, PCT and IL-6 detection simultaneously utilizing three peptides (CSKSK, CYR and CRYR) in the following experiments.

respectively (Fig. S2A, D and G). We also use the concentration of different peptides (60, 60 and 80 µM for CSKSK, CYR and CRYR respectively) that cause the maximum decrease of A600/A530 for further assays (Fig. S2B, E and H). Finally, we prepare the different sizes of AuNPs, including 13 nm, 30 nm and 60 nm (Fig. S1) to explore the size effects (Fig. S2C, F and I) and employ a constant size of AuNPs (30 nm) of all three assays based on CSKSK, CYR or CRYR for detection, taking both the sensitivity and stability into consideration for naked eye-detection.

Figure 4. The screening experiment of different peptides through evaluating their analytical performances for ALP sensing. (A) The photos and the absorbance spectra of AuNPs containing CSKSK treated with [ALP] from 0 to 150 U/L. (B) The photos and the absorbance spectra AuNPs containing CYR treated with [ALP] from 0 to 75 U/L. (C) The photos and the absorbance spectra of AuNPs containing CRYR treated with [ALP] from 0 to 37.5 U/L. (D) The photos and the absorbance spectra of AuNPs containing CYK treated with [ALP] from 0 to 150 U/L.

Figure 5. The characterization for the aggregation of AuNPs. (A) The hydrodynamic diameter of AuNPs after the addition of ALP-treated/untreated peptides by DLS. The insets are the corresponding photographs of AuNPs solutions. (B) TEM images of AuNPs after the addition of the untreated peptides. (C) TEM images of AuNPs after the addition of ALP-treated peptides 1 (CSKSK). (D) TEM images of AuNPs after the addition of ALP-treated peptides 2 (CYR). (E) TEM images of AuNPs after the addition of ALP-treated peptides 3 (CRYR).

The characterization of AuNPs. We utilize both transmission electron microscopy (TEM) and dynamic light scattering (DLS) to characterize the aggregation of AuNPs (Fig. 5). At a same concentration for ALP (37.5 U/L), the hydrodynamic diameter of AuNPs increases differently from 30 nm to around 100, 514 and 2000 nm in the presence of ALP -treated CSKSK, CYR and CRYR (Fig. 5a) respectively. We confirm the morphology and size distribution of the AuNPs by TEM. AuNPs are well dispersed in the absence of ALP but aggregate upon the addition of mixtures of ALP (37.5 U/L) and three peptides (60 µmol/L) (Fig. 5B-E). Besides the size distribution, the structures of the AuNPs are different after the addition different types peptides (Fig. 5C-E), which coincides the spectrum study on peptide-sensitive aggregation of AuNPs (Fig. 5A), and justifies the possibility of using these different peptides for sensing multiple biomarkers with broadly ranged concentrations. Optimizing the condition of peptide-ALP-AuNPs (PAA) reaction We optimize the reaction conditions of PAA reaction including reaction time, the [peptide] and the sizes of AuNPs. After adding different kinds of ALP-treated peptides, the initial absorbance peak (522 nm) of AuNPs gradually decreases with a small red shift (530 nm), and the emerging peak at 600 nm. In all three assays based on CSKSK, CYR or CRYR, the A600/A530 values decrease gradually and remain constant after a sufficient period of time, and we choose 60, 40 and 60 min for these assays

The Sensitivity of PAAI for CRP, PCT and IL-6 Detection. We test the sensitivity of our assay toward three biomarkers. PCT, IL-6 and CRP are inflammatory biomarkers in clinics, and simultaneous detection of these biomarkers using the same assay can dramatically enhance the accuracy of diagnosis and increase detection efficiency. The concentration of PCT and IL-6 in human serum is low (typically at pg/mL level) while that of CRP is at µg/mL level, which requires the diagnostic method not only has high sensitivity but also has broad detection range from pg/mL to ng/mL.47-48 In clinics, CRP is a biomarker for diagnosis of infection and inflammation diseases, whose blood concentration in a normal person is between 1 to 10 µg/mL.49 CRP concentration higher than 10 µg/mL may indicates different diseases such as inflammation, viral infection (10-40 µg/mL), or bacterial infection (40-200 µg/mL).50 We conduct a typical sandwich procedure for CRP detection using PAAI with CSKSK (Fig. 1B). In the absence of CRP, the ALP that linked to detection antibody

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will not present on the surface of plates during the sandwich procedures, and the solution color of AuNPs would remain red. In contrast, in the presence of CRP, AuNPs would aggregate as a result of ALP-dephosphorylated peptide, and a color change from red to blue that can be observed with naked eye rapidly. The color change compared to blank sample is distinguishable when the concentration of CRP is 3.15 µg/mL, which indicates that our method can detect 3.15 µg/mL of CRP by naked eyes. The absorbance peak of AuNPs solution gradually shifts from 530 nm to 600 nm with the increasing concentrations of CRP from 0 to 100 µg/mL (Fig. 6A), and we observe a linear relationship between the A600/A530 ratio and the log concentration of CRP (3.15 µg/mL-100 µg/mL) (Fig. 6B), the linear relation equation is Y=0.43X+0.32, R2=0.97, and the LOD based on UV-vis measurement is 1.15 µg/mL. We use PAAI with CRYR for the detection of PCT. PCT is an important and specific biomarker for diagnosis of infection and inflammation diseases, whose blood concentration in a normal person is under 0.5 ng/mL.51 We perform PCT detection using PAAI with CYR using similar procedure to that of CRP. Such method can detect 5 ng/mL of PCT by naked eyes, and we observe a linear relationship between the A600/A530 ratio and the log concentration of PCT (0.2 ng/mL-25 ng/mL) (Fig. 6C and D), the linear relation equation is Y=0.36X+0.65, R2=0.98, and the LOD based on UV-vis measurement is 0.24 ng/mL. We use PAAI with CRYR for IL-6 detection, since IL-6 is also an important infection biomarker, the concentration of IL-6 in health people is very low (under 0.1 ng/mL), thus, it needs highly sensitive assays such as Roche electrochemiluminescent immunoassay (REI) for detection of IL-6. Using similar procedure,52 we can detect 50 pg/mL of IL-6 by naked eyes and observe a linear relationship between the A600/A530 ratio and the IL-6 concentration (50 pg/mL-1600 pg/mL) (Fig. 6E and F), the linear relation equation is Y=0.0048X+0.38, R2=0.98, and the LOD based on UV-vis measurement is 12.51 pg/mL. We compare the analytical performances of PAAI for detection of CRP, PCT and IL-6 with those of published reports, and they are similar(Table S2).48-55 Compared to other AuNPs-based immunoassays, this PAAI has several advantages: i) We can adjust the sensitivity of the PAAI through changing the structures of the designed short peptides when we apply it to detect different kinds of targets, which provides an effective and straightforward strategy for simultaneous detection of different but clinically relevant biomarkers with broad linear range; ii) Through the introduction of the enzymatic reaction between peptide and ALP, AuNPs require no additional surface modification, providing a straightforward and convenient assay process; iii) The PAAI can accommodate commercial ELISA because ALP is a stable, highly efficient and commercialized labeling enzyme, and PAAI has the comparable cost with commercial ELISA kit, about at $2 per sample. The specificity and reproducibility of PAAI. To evaluate the anti-interference property of the PAAI system, we conduct the specificity experiment toward CRP, PCT and IL-6 respectively. We investigate the specificity of the PAAI for detection of CRP by employing Human IgG (5 µg/mL), PCT (80 ng/mL) and IL-6 (100 pg/mL) as interferences and perform PAAI under the same experimental conditions. None of these three interferences would induce the color

change of AuNPs in our system even at a high concentration. After mixed with CRP (50 µg/mL), the values of A600/A530 from these samples exhibit no significant difference when compared with samples containing only CRP (Fig. S3A). Under the same procedure, other interferences would not influence our assays toward CRP (Fig. S3B) or IL-6 (Fig. S3C) detection using CYR or CRYR-based assays. This result suggests the high selectivity and anti-interference property of our method for further clinical diagnostics. We also evaluate the stability and variations of PAAI system, the intra coefficient of variation (CV) and inter CV are both below 15%, which indicates the good stability and repeatability of PAAI (Table S3).

Figure 6. The sensitivity of PAAI for CRP, PCT and IL-6 detection. (A, B) The color, spectrum change and corresponding relationship between the concentration of CRP (0 to 100 µg/mL) and the A600/A530 value using PAAI with CSKSK. (C, D) The color, spectrum change and corresponding relationship between the concentration of PCT (0 to 100 ng/mL) and the A600/A530 using PAAI with CYR. (E, F) The color, spectrum change and corresponding relationship between the concentration of CRP (0 to 1600 pg/mL) and the A600/A530 value using PAAI with CRYR. The insets are the corresponding photographs of AuNPs solutions. Detection of CRP, PCT and IL-6 in clinical serum samples. We explore the practicality of PAAI in clinical samples and compare it with Roche ECL method and ELISA. We receive clinical serum samples from 15 patients who possibly suffer from infectious diseases, and we simultaneously detect the CRP, PCT and IL-6 in these samples. The simultaneous detection of these three markers in clinical samples can help improve the accuracy of

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diagnosis and reduce the abuse of antibiotics.6,56 For clinical serum sample analysis, we divide one serum sample into three parts in three wells of 96 well plate, and every part is used to detect one target. We only need to select the suitable peptide according to the requirement of targets following the same protocol in this PAAI, which avoids the disadvantages of sample preparation shows great potential for developing automated and convenient detection for multiple targets. For comparison, we also use conventional methods routinely employed in clinics to obtain the quantitative results for detection of CRP (ELISA) and PCT and IL-6 (Roche-ECL) in the same set of real serum samples. (Table S4). There is no significant difference between the results that obtained from these two different methods, and the signal obtained from PAAI can be read out more easily by naked-eyes due to the obvious color change (red-to-blue), indicating that our approach holds great promise for the simultaneous detection of these infection markers with convenient operation. The comparison between PAAI, ELISA and Roche ECL for quantification of CRP, PCT and IL-6 showed a good agreement of two methods (Fig. 7A, B and C), suggesting that the PAAI is reliable for the multiple detections of clinical biomarkers. Compared with Roche ECL, the PAAI is a point-of-care testing (POCT) assay, which can provide an attractive tool in poverty-stricken area with low cost and good conveniences. To justify the potential of our system in conducting portable devices, we perform automated signal readout through RGB analysis of the photos of the final AuNPs solutions with the color scanner APP from smartphone (Fig. S4), and we employ the blue value of the mixtures to enable the quantification of different biomarkers. The results from these two signal readout strategies have acceptable correlation, which makes this assay more suitable for POCT in clinical diagnosis.

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The comparison between PAAI and ELISA for quantification of CRP. (B) The comparison between PAAI and Roche ECL method for quantification of PCT. (C) The comparison between PAAI and Roche ECL method for quantification of IL-6.

CONCLUSION In this work, we develop a convenient colorimetric immunoassay with tunable detection range by utilizing peptide-mediated modulation of AuNPs. This approach requires no additional surface modification of AuNPs, and allows for detecting multiplex targets (both low-abundance and high-abundance) by naked eyes with a broad detection range. Our research provides a large chemical space for developing controllable immunoassays by the employment of peptides. Further work we would focus on integrating our assay with solid form assays, such as paper-based device, or microfluidics chips to enable more effective and straightforward assays for the needs of POCT in clinical diagnosis, food safety, environmental monitoring.

ASSOCIATED CONTENT Supporting Information Additional experimental data, including the potential of the original peptides, the quantitative results of PAAI and Roche-ECL method for detection of PCT and IL-6 in real serum samples, the quantitative results of PAAI and ELISA method for detection of CRP and the peptides after enzyme treatment, the optimization of our assay, The selectivity of the PAAI for PCT, CRP and IL-6 detection. Figure S1-S3 Table S1-S2

AUTHOR INFORMATION Corresponding Authors [email protected] (YP Chen) [email protected] (ZY Qian) [email protected] (XY Jiang) Notes The authors declare no competing financial interests. #These

authors contribute this work equally

ACKNOWLEDGMENT We thank the National Science Foundation of China (81671784, 21505027, 81361140345, 21535001, 81730051, 21761142006), Beijing Nova Program (Z181100006218017), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2018047), Chinese Academy of Sciences (XDA09030305, 121D11KYSB20170026), the National Natural Science Fund for Distinguished Young Scholars (NSFC31525009) and Sichuan Innovative Research Team Program for Young Scientists (2016TD0004) for financial support.

REFERENCES (1) Chen, Y; Xianyu, Y.; Jiang, X. Acc. Chem. Res. 2017, 50, 310-319. (2) Zhang, Y.; Guo, Y.; Xianyu, Y.; Chen, W.; Zhao, Y.; Jiang, X. Adv. Mater. 2013, 25, 3802-3819.

Figure 7. The PAAI, ELISA and Roche ECL method for detection of CRP, PCT and IL-6 in real clinical samples. (A)

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For TOC use only

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