Multiple SNPs Detection Based on Lateral Flow Assay for

Feb 16, 2018 - Furthermore, this ARMS-LFA system was applied to clinical trials for screening the seven pathogenic SNPs in PAH of 23 families includin...
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Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

Multiple SNPs Detection Based on Lateral Flow Assay for Phenylketonuria Diagnostic Xiaonan Liu,†,⊥ Chao Zhang,†,⊥ Kewu Liu,† Han Wang,‡ Chaoxia Lu,‡ Hang Li,† Kai Hua,†,§ Juanli Zhu,∥ Wenli Hui,†,§ Yali Cui,*,†,§,∥ and Xue Zhang*,‡ †

College of Life Sciences, Northwest University, Xi’an, Shaanxi 710069, China McKusick-Zhang Center for Genetic Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China § National Engineering Research Center for Miniaturized Detection System, Xi’an, Shaanxi 710069, China ∥ Shaanxi Provincial Engineering Research Center for Nano-Biomedical Detection, Xi’an, Shaanxi 710077, China ‡

S Supporting Information *

ABSTRACT: Single nucleotide polymorphisms (SNPs) are closely related to genetic diseases, but current SNP detection methods, such as DNA microarrays that include tedious procedures and expensive, sophisticated instruments, are unable to perform rapid SNPs detection in clinical practice, especially for those multiple SNPs related to genetic diseases. In this study, we report a sensitive, low cost, and easy-to-use point-of-care testing (POCT) system formed by combining amplification refractory mutation system (ARMS) polymerase chain reaction with gold magnetic nanoparticles (GMNPs) and lateral flow assay (LFA) noted as the ARMS-LFA system, which allow us to use a uniform condition for multiple SNPs detection simultaneously. The genotyping results can be explained by a magnetic reader automatically or through visual interpretation according to the captured GMNPs probes on the test and control lines of the LFA device. The high sensitivity (the detection limit of 0.04 pg/μL with plasmid) and specificity of this testing system were found through genotyping seven pathogenic SNPs in phenylalanine hydroxylase gene (PAH, the etiological factor of phenylketonuria). This system can also be applied in DNA quantification with a linear range from 0.02 to 2 pg/μL of plasmid. Furthermore, this ARMS-LFA system was applied to clinical trials for screening the seven pathogenic SNPs in PAH of 23 families including 69 individuals. The concordance rate of the genotyping results detected by the ARMS-LFA system was up to 97.8% compared with the DNA sequencing results. This method is a very promising POCT in the detection of multiple SNPs caused by genetic diseases.

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rather than direct detection of specifically known SNPs. Other technologies that rely on sample amplification, including DNA microarrays,10−12 real-time polymerase chain reaction (RTPCR),13,14 self-sustained sequence replication (3SR),15 strand displacement amplification (SDA),16 high resolution melting (HRM),17 Mass ARRAY,18,19 the multiplex PCR-RFLP method,20 and isothermal DNA amplification,21,22 also offer sensitive methods to detect pathogenic SNPs. However, tedious experimental procedures and expensive and sophisticated instruments limit the application of these techniques in multiple pathogenic SNPs detection.23,24 The tremendous potential of genetic testing in clinical practice desperately demands efficient screening methods which meet the requirements of point-of-care testing (POCT) strategies.25 In order to

ith a high-quality reference sequence of the human genome now available based on the completion of the human genome project,1 attention is now rapidly shifting toward the study of individual genetic variations. The most abundant source of genetic variation (approximately 90%) in the human genome is represented by single nucleotide polymorphisms (SNPs), which account for heritable interindividual differences in complex phenotypes.2,3 SNPs which are related to genetic disease have a great potential for direct clinical application by providing highly accurate diagnostic information for facilitating early diagnostic, prevention, and treatment of genetic diseases.4,5 By virtue of accuracy, DNA sequencing is considered as the gold standard for SNPs detection. With the development of next-generation sequencing (NGS) technology, it is widely used in the sequencing of human genomes to look for pathogenic SNPs.6 However, as per the theory of the methodology and the subsequent massive data analysis, sequencing is more suitable for discovery of novel SNPs7−9 © XXXX American Chemical Society

Received: December 8, 2017 Accepted: February 9, 2018

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DOI: 10.1021/acs.analchem.7b05113 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry satisfy the application of SNPs detection in clinical practice, it would be interesting to simplify the procedure of genotyping. Gold magnetic nanoparticles (GMNPs) are composite particles with significantly elevated stableness and compatibility for biological interactions.26 This composite nanomaterial shows great potential serving as diagnostic or therapeutic agents in a variety of medical fields.27−30 In a previous study, we established a lateral flow assay (LFA) system assembled with GMNPs that relies on the immune hybridization reaction as one of the POCT formats, which is affordable, user-friendly, rapid, robust, and scalable for nucleic acid analysis.28,31 However, the application of LFA in screening of genetic diseases with multiple SNPs is made difficult by different detection conditions for gene mutations. In this study, we demonstrated a multichannel POCT system formed combining amplification refractory mutation system (ARMS) polymerase chain reaction with GMNPs and LFA noted as the ARMS-LFA system, which enables us to use a uniform condition for multiple SNPs detection simultaneously. The genotyping result can be explained through a magnetic reader automatically or be visually interpreted according to the captured GMNPs probes on the test and control lines of the LFA device, and more importantly, complicated operation steps and complex data analysis are avoided in this system32,33 with no need for expensive and sophisticated instruments. The high sensitivity and specificity of the ARMS-LFA system were found by genotyping seven pathogenic SNPs (R111X, IVS4-1, Y204C, R243Q, W326X, Y356X, and R413P) in the phenylalanine hydroxylase gene (PAH)34−38 which lead to phenylketonuria (PKU), a typical neonatal hereditary disease including multiple pathogenic SNPs.39,40 Furthermore, our ARMS-LFA system was applied to clinical trials for screening the seven pathogenic SNPs in PAH of 23 families including 69 individuals. The concordance rate of the genotyping results detected by the ARMS-LFA system was up to 97.8% compared with the DNA sequencing results, which make this method a very promising POCT in the detection of multiple SNPs-caused genetic diseases.

glycosylase (UDG) were obtained from Shinegene Molecular Biotechnology Co., Ltd. (Shanghai, China). Plasmids Construction and Identification. Plasmids containing wild type and homozygous mutation fragments of the seven pathogenic SNPs (R111X, IVS4-1, Y204C, R243Q, W326X, Y356X, and R413P) in PAH were constructed using the pMD19-T Vector Cloning Kit (Takara Bio Inc. Dalian, China) with site-directed mutagenesis primers (Table S-1) and were extracted from transformed Escherichia coli DH5α cells using a TIAN prep Mini Plasmid Kit (Tiangen Biotech Co., Ltd. Beijing, China). All plasmids were identified via sequencing by Beijing Genomic Institute (BGI, Beijing, China; Figure S-1). The concentration of plasmids was determined using a NanoDrop 2000c/2000 UV−vis spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, U.S.A.). Establishment and Optimization of the ARMS-LFA System. GMNPs was synthesized according to the methods described previously.26,41 The GMNPs were functionalized with cetyltrimethylammonium bromide (CTAB) surfactant, followed by modification of poly acrylic acid (PAA) and conjugation of anti-digoxin antibody through 1-[3(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride chemistry according to a previous report.42,43 The LFA is performed over a strip, which is composed of five overlapping pads, i.e., sample pad, conjugate pad, nitrocellulose membrane, absorbent pad, and plastic cushion. A schematic diagram of the lateral flow strip is shown in Figure 1A. Briefly, streptavidin and goat anti-mouse IgG were preimmobilized respectively via BioJet HM3010 dispenser (BioDot Inc., California, U.S.A.) in a defined test line (T line) and a control line (C line) on a porous nitrocellulose membrane. Then, the solution containing anti-digoxin antibody conjugated GMNPs (GMNPs-anti-Dig)

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EXPERIMENTAL SECTION Oligonucleotides and Reagents. For convenience, we refer to the wild-type as “WT” and the mutation type as “M”. To genotype seven pathogenic SNPs (R111X, IVS4-1, Y204C, R243Q, W326X, Y356X, and R413P) in PAH, a set of three specific primers were designed for each allele using the Primer 5.0 software program (Primer-E Ltd., Plymouth, U.K.), including a 5′ biotin labeled common primer, a 5′ digoxin labeled specific primer for WT, and a 5′ digoxin labeled specific primer for M. Two primers were also designed for sequencing. The primers sequences are shown in Table S-1. All primers were synthesized by Invitrogen Biotechnology Ltd. (Shanghai, China). All chemicals were of analytical grade and purchased from reputable vendors. Buffers were prepared according to standard laboratory procedures. Anti-digoxin antibody was purchased from Meridian Life Science, Inc. (Saco, ME, U.S.A.). Streptavidin was obtained from Promega Biotech Inc. (Madison, WI, U.S.A.). Goat anti-mouse IgG was from Joey Bioscience Inc. (Shanghai, China). HotMaster Taq DNA Polymerase and 10× PCR buffer were purchased from TIANGEN Biotech Co., Ltd. (Beijing, China). dNTPs (including dATP, dUTP, dCTP, and dGTP) and uracil-DNA

Figure 1. Schematic diagram of the ARMS-LFA system. (A) Structure of labeled lateral flow device. (B) Sample preparation and target amplification. (C) Result analyzed based on the signal read-out by a magnetic reader automatically or by visual interpretation. (D) The genotyping results of the seven SNPs in PAH. B

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Analytical Chemistry Table 1. Optimized Primer Concentrations of the ARMS-LFA System allele primer

R111X

IVS4-1

Y204C

R243Q

W326X

Y356X

R413P

mutation primer set wild type primer set

30 nM 30 nM

20 nM 20 nM

30 nM 30 nM

30 nM 100 nM

60 nM 10 nM

50 nM 30 nM

100 nM 20 nM

primers (corresponding WT and M) are added into two tubes for PCR, respectively. In order to improve the specific amplification of the targeted gene, the allele-specific primers contain an additional mismatch at the penultimate 3′ nucleotide to increase the strength of allelic discrimination according to the principle of ARMS44−46 and mismatch amplification mutation assay.47,48 The target PCR products will be acquired only when the 3′-end of the specific primer is completely complementary with the template. As shown in Figure 1C, by addition of the PCR products on sample pads of lateral flow strips after amplification, digoxin labeled on the 5′-end of specific primers can be recognized and captured by anti-digoxin antibody coated GMNPs (GMNPsanti-Dig) on the conjugate pad. Driven by capillary force, the PCR products-GMNPs composites migrate along the strip and aggregate on the test line (T line) through conjugation between T line immobilized streptavidin and biotin labeled on the 5′end of common primers, which make the T line a red band due to the bathochromic effect of GMNPs. The rest of the GMNPsanti-Dig keep moving and are captured by goat anti-mouse IgG on the control line (C line), which present another red band that confirms the efficacy of the lateral flow system. In the absence of target PCR products, no red band is observed on the T line. The result can be read by using a magnetic reader to obtain the magnetic signal on the T line and C line or visually read-out according to the coloration of the T line. As shown in Figure 1D, the genotyping results in PAH can be visually interpreted without the need of any instruments. A final genotyping result of an allele was visually interpreted as per the color development on the T lines of both strips (Figure 2A). For the homozygous mutant allele, a distinct red band was observable on the T line of the strip used only for the M tube but not for the WT tube. In contrast, for the wild type

was dispensed on the conjugate pad. The strips were dried and stored in a sealed aluminum foil bag at room temperature. For each SNP detected, two separate PCR reactions (M tube and WT tube) were run simultaneously using the same template. All PCR product was dropped onto the sample pad after amplification for each reaction tube. The reference plasmid samples (confirmed by sequencing) were used to validate the method. The final reaction volume of PCR was 50 μL, including 10× reaction buffer (10 mM Tris-HCl and 50 mM KCl), 0.2 mM of each dNTPs (dATP, dUTP, dCTP, and dGTP), 3 mM MgCl2, 0.5 U of Hotmaster Taq DNA polymerase, 0.5 U of UDG polymerase, a certain concentration (Table 1) of primer set including common primer and allelespecific primer (M primer in M tube and WT primer in WT tube), and 3 μL of prepared genomic DNA. All of the amplifications were performed by using a 2720 Thermal Cycler (Applied Biosystems, Foster City, U.S.A.) with the following parameters: two initial denaturation steps for 2 min at 50 °C and 5 min at 94 °C, 30 cycles of 30 s at 94 °C, 30 s at 60 °C and 30 s at 65 °C, and one step of 10 min at 65 °C. To obtain the optimal performance, specific cycle number (29, 30, 31, 32, and 33 cycle), annealing temperature (58, 59, 60, 61, and 62 °C) , and concentration of primers (10, 20, 30, 40, 50, 60, 100, 150, and 200 nM) were evaluated with ARMS-LFA system. Evaluation of the ARMS-LFA System. The sensitivity of the optimized ARMS-LFA system was determined by using a gradient dilution series of plasmid. The magnetic signal at the T line and C line of the strips was determined by using a magnetic reader (MagnaBioScience LLC, U.S.A.). Moreover, we also evaluated the reproducibility of this system with three batches of LFA strips. Clinical Application of the ARMS-LFA System. Fresh whole blood samples were collected from 23 families including 69 individuals (23 PKU child patients and their parents) in EDTA anticoagulant tubes at the Peking Union Medical College Hospital (Beijing, China) with informed consent. The study was approved by the ethic committee of the College of Life Sciences, Northwest University (Xi’an, China). All methods were performed in accordance with the approved guidelines. The genotype of each sample was analyzed by the ARMS-LFA system and compared with the results provided by sequencing in BGI with sequencing primers (Table S-1). Based on the statistics data, the coincidence rate of each allele and total agreements were calculated to evaluate the accuracy of our method.

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RESULTS AND DISCUSSION Principle of the ARMS-LFA System. The schematic of the general sandwich format of LFA is shown in Figure 1A. The LFA is performed over a strip, which is composed of five overlapping pads, i.e., sample pad, conjugate pad, nitrocellulose membrane, absorbent pad, and plastic cushion. As exhibited in Figure 1B, genomic DNA was purified from whole blood sample followed by amplification through PCR with specifically designed primers. For each SNP in PAH, two sets of primer including common primer and allele-specific

Figure 2. Genotyping result (take R111X as an example) provided by (A) the ARMS-LFA system through visualized interpretation, (B) the ARMS-LFA system through magnetic reader automatically, (C) agarose gel electrophoresis, and (D) DNA sequencing, respectively. C

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Analytical Chemistry allele, the red band showed exclusively on the strip receiving the WT tube but not the M tube. However, when red bands with similar intensities were present on the T lines of both strips, it indicated a heterozygous allele. In addition, the genotyping result can also be explained by using a magnetic reader to detected the magnetic signal on the T line and C line (Figure 2B). The magnetic signal on the T line will be acquired only when the red band is observable accompanied by the target PCR products. Furthermore, the genotyping results detected by agarose gel electrophoresis (Figure 2C) and DNA sequencing (Figure 2D) were displayed as a comparison, which indicated the same accuracy of the ARMS-LFA system as a gold standard of genotyping methods. Optimization of the ARMS-LFA System. In order to obtain optimal experimental conditions, we examined the performance of the ARMS-LFA system under different cycle numbers, annealing temperatures, and concentrations of primers. The magnetic signal peak value and brightness at the T line shows the best amplification efficiency and specificity when the cycle number was 30 (Figure S-2 and Table S-2) and the annealing temperature was 60 °C (Figure S-3 and Table S3). In addition, the best primer concentrations for different SNPs are displayed in Table 1 (Figure S-4 and Table S-4). Performance of the ARMS-LFA System. The sensitivity of the ARMS-LFA system was determined with plasmid containing homozygous mutation genotype of R111X in PAH. A series of reaction mixtures containing plasmid at different final concentrations from 0.02 to 2 pg/μL were run with our system. The magnetic signals at T and C lines were detected using a magnetic reader automatically. The plasmid concentration as low as 0.04 pg/μL still provides a positive result obtained by visually read-out or using a magnetic reader indicating a high sensitivity of this system with a detection limit of 0.04 pg/μL (Figure 3A, B). The PCR amplification efficiency (presented by relative magnetic unites (RMU)) significantly improved as the concentration of the plasmid increased (Figure 3C). Moreover, as shown in Figure 3D, a standard curve was plotted with the average of magnetic signals at the T line against plasmid concentrations. The linear range from 0.02 to 2 pg/μL was found with the standard curve. The linear regression

equation (y = 274.65x + 4.104) with high correlation coefficient (R2 > 0.997) indicated that the ARMS-LFA system can be applied in DNA quantification. The reproducibility of the ARMS-LFA system was assessed by analyzing plasmids with wild type, heterozygous mutation, and homozygous mutation genotypes of the seven pathogenic SNPs, respectively. Each sample was run with the ARMS-LFA system in triplicate with different batches of strips and followed through visual detection and with a magnetic reader. The results presented in Figure S-5 and Table S-5 indicate that no significant distinction was obtained among the three pairs strips. Clinical Application of the ARMS-LFA System. To evaluate the reliability of the optimized ARMS-LFA system in this study, the accuracy of this system was further verified by clinical samples (whole blood from 23 families including 69 individuals). By genotyping the seven SNPs for PAH (R111X, IVS4-1, Y204C, R243Q, W326X, Y356X, and R413P) of 69 individuals with the ARMS-LFA system, 92 mutant alleles were found. As shown in Figure 4A, the genotyping results in PAH in

Figure 4. (A) Genotyping result of the clinical sample and (B) frequency of different gene mutations in PAH.

23 families were in accordance with the recessive genetic disease inheritance. The frequencies of different mutant alleles are displayed in Figure 4B, and the frequencies of mutant alleles in PAH provided with ARMS-LFA system were in accordance with those reported by Zhu and Song et al.37,38 in the Chinese population. As shown in Table 2, a concordance rate up to 97.8% was found with the ARMS-LFA system compared with DNA sequencing, which clearly demonstrates that our system is comparable to the DNA sequencing in the seven pathogenic SNPs in PAH genotyping. Here we have established a sensitive, low cost, and easy-touse SNPs detection platform using ARMS-LFA, which enables us to detect multiple SNPs of specific genetic diseases under uniform conditions simultaneously. With the help of gold magnetic nanoparticles, genotyping results can be provided both by using a magnetic reader automatically and visual inspection of colors on the T and C lines quantificationally and qualitatively. The major advantage of this assay is a rapid qualitative answer in “‘yes’” or “‘no’” terms. Compared with conventional PCR-based SNPs detection methods or DNA sequencing, our assay shows significant advantages.49,50 More specifically, the following advantsages have been found. (a) By using ARMSLFA system, it takes only 5 min to obtain the detection results without the need for expensive or high-end instruments. (b) Existing genotyping methods always require different conditions for multiple SNPs detection, which will inevitably increase the reaction time and labor output, whereas a uniform condition is sufficient for our testing system. (c) Previous techniques are usually run with a complex operation procedure

Figure 3. (A) Brightness of the T and C lines at different concentrations of plasmid. (B) Curve graph of the magnetic signal against plasmid concentrations. (C) Magnetic signal peak value of the T and C lines at different concentrations of plasmid. (D) The standard curve of the magnetic signals of the T line against plasmid concentrations. D

DOI: 10.1021/acs.analchem.7b05113 Anal. Chem. XXXX, XXX, XXX−XXX

total

heterozygous mutation

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

homozygous mutation

R111X IVS4-1 Y204C R243Q W326X Y356X R413P others R111X IVS4-1 Y204C R243Q W326X Y356X R413P others

R111X

sequencing (n = 69) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

IVS4-1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1

R243Q 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

W326X

homozygous mutation Y204C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Y356X 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

R413P 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

others 0 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 10

R111X

ARMS-LFA (n = 69)

0 0 0 0 0 0 0 0 0 8 0 0 0 0 0 0 8

IVS4-1 0 0 0 0 0 0 0 0 0 0 8 0 0 0 0 0 8

Y204C 0 0 0 0 0 0 0 0 0 0 0 24 0 0 0 0 24

R243Q 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

W326X

heterozygous mutation 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 4

Y356X 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 2

R413P

Table 2. Genotyping Results of 23 Families Including 69 Individuals Containing 92 Gene Mutations for the Seven Pathogenic SNPs in PAH

0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 32 34

others 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 2

discrepant

0 0 0 1 0 0 0 0 10 8 8 26 0 4 2 32 0

total

100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 92.31% 100% 100% 100% 100% 97.80%

agreement

Analytical Chemistry Article

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Analytical Chemistry

Founds (No. 2017KCT-25), and Northwest University Graduate Innovation and Creativity Funds (No. YZZ15008).

as well as expensive and sophisticated instruments that may not be available in many laboratories, whereas the present methods provide an easy-to-operate and affordable on-site technique for genotyping with high efficiency. Therefore, this method can be used in the laboratories of all levels of hospitals and medical institutions, especially for laboratories with limited resources. The ARMS-LFA system established for the pathogenic SNPs in PAH genotyping with high specificity and sensitivity can be extended to the genotyping of other multiple SNPs causative genetic diseases, which making it a valuable molecular diagnostic tool and useful in clinical practice.

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CONCLUSIONS In this study, we presented the ARMS-LFA system to detect multiple SNPs of specific genetic diseases under uniform conditions. This is the first report of a multiple SNPs detection system combining ARMS with GMNPs and LFA for genotyping. Our data has demonstrated that the system is highly applicable for the detection of multiple SNPs in clinical samples due to its various advantages, such as short detection time, simple procedure, and superior sensitivity and specificity. This newly established ARMS-LFA system can be further adapted for the detection of other genes with multiple SNPs that are associated with disease risk, drug metabolism, or drug reaction.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b05113. Results of DNA sequencing of all constructed plasmids. The magnetic signal peak value and brightness of the T line at different cycle numbers, annealing temperatures, and primer concentrations. The genotyping results of the ARMS-LFA system with three batches of strips. Primer sequences used in the ARMS-LFA system for PAH genotyping. The relative standard deviation of reproducibility. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +86 13501362561. Fax: +86-10-69155110. *E-mail: [email protected]. Tel: +86 29-88302383. Fax: +8629-88303551. ORCID

Yali Cui: 0000-0001-8397-4588 Author Contributions ⊥

X.L. and C.Z. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (No. 81772289), the National Key Research and Development Program of China (No. 2016YFC0905100), the CAMS Innovation Fund for Medical Sciences (CIFMS; No. 2016-I2M-1-002), the National Natural Science Foundation of China (No. 31771083), Shaanxi Provincial Nano-Biomedical Detection Innovation Team F

DOI: 10.1021/acs.analchem.7b05113 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.7b05113 Anal. Chem. XXXX, XXX, XXX−XXX