Polyelectrolyte-Coated Gold Magnetic Nanoparticles for Immunoassay

Subscriber access provided by University of Leicester

Article

Polyelectrolyte Coated Gold Magnetic Nanoparticles for Immunoassay Development: Toward Point of Care Diagnostics for Syphilis Screening Dong Yang, Jianzhong Ma, Qinlu Zhang, Ningning Li, Jiangcun Yang, Paul Ananda Raju, Mingli Peng, Yanling Luo, Wenli Hui, Chao Chen, and Yali Cui Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac400517e • Publication Date (Web): 04 Jun 2013 Downloaded from http://pubs.acs.org on June 9, 2013

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Polyelectrolyte Coated Gold Magnetic Nanoparticles for Immunoassay Development: Toward Point of Care Diagnostics for Syphilis Screening ‖

‖

‖

Dong Yang†, , Jianzhong Ma*,‡, Qinlu Zhang , Ningning Li , Jiangcun Yang #, Paul Ananda ‖ ‖ ‖ ‖ ‖ Raju , Mingli Peng , Yanling Luo , Wenli Hui§, Chao Chen§, and Yali Cui*,§, †

College of Chemistry and Chemical Engineering, Shaanxi University of Science and Technology,

Xi’an, 710021, China ‡

College of Resource and Environment, Shaanxi University of Science and Technology, Xi’an,

710021, China §

College of Life Sciences, Northwest University, Xi’an, 710069, China

#

Shaanxi Provincial People’s Hospital, Xi’an, 710068, China

‖

National Engineering Research Center for Miniaturized Detection Systems, Xi’an, 710069,

China

Corresponding authors: *

Tel: 0086-29-88302383. Fax: 0086-29-88303551. E-mail: [email protected]

*

Tel: 0086-29-86168010. Fax: 0086-29-86168012. E-mail: [email protected]

ABSTRACT: Immediate response for disease control relies on simple, inexpensive and sensitive diagnostic tests, highly sought after for timely and accurate test of various diseases including infectious diseases. Composite Fe3O4/Au nanoparticles have attracted considerable interest in diagnostic applications due to their unique physical and chemical properties. Here, we developed a simple coating procedure for gold magnetic nanoparticles (GMNs) with poly (acrylic acid) (PAA). PAA coated GMNs (PGMNs) were stable and mono-dispersed, and characterized by Fourier transform-infrared spectroscopy (FT-IR), transmission electron microscope (TEM), UV-visible scanning spectrophotometer (UV-Vis), thermo gravimetric analyzer (TGA) and Zetasizer methodologies. For diagnostic application, we established a novel lateral flow immunoassay (LFIA) strip test system where recombinant Treponema pallidum antigens (r-Tp) were conjugated with PGMNs to construct a particle probe for detection of anti-Tp antibodies. Intriguingly, the particle probes specifically identified Tp antibodies with a detection limitation as 1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

low as 1 national clinical unit/mL (NCU/mL). An ample pool of 1,020 of sera samples from three independent hospitals were obtained to assess our PGMNs-based LFIA strips, which exhibited substantially high values of sensitivity and specificity for all clinical tests (higher than 97%), and therefore proved to be a suitable approach for syphilis screening at a point-of-care test (POCT) manner. KEYWORDS: Gold magnetic nanoparticles (GMNs), Poly (acrylate acid) (PAA), Lateral flow immunoassay (LFIA), Treponema pallidum (Tp), Point-of-care test (POCT). ■ INTRODUCTION Viable point-of-care testing (POCT) aims to provide conveniently and immediately medical testing at or near the site patient. The practice of POCT gradually applies in the fields of diagnostic and disease screening, and is thought to ultimately attain a significant positive impact via its cost-effective attribute in health care system. Superparamagetism, optical properties and surface chemistry have made gold magnetic nanoparticles (GMNs) versatile for a broad range of biomedical applications including drug/gene delivery,1-4 magnetic separation,5-8 magnetic resonance imaging (MRI),9-10 and biosensing.11-13 In the emerging and revolutionary diagnostic arena of POCT, GMNs-based immunoassays, tailored to be simple, fast, ultrasensitive and portably readable, can potentially provide “instantaneous” diagnosis near the patients (e.g. hospital bed and home), circumventing the need for a clinical laboratory. The structure of GMNs comprises an iron core (Fe3O4) and a layer of gold deposited on its surface – superparamagetism emanates from the iron core whereas optical properties and surface chemistry are attributes of gold.14 The surface chemistry of gold, relatively inert, and subsequently stable and biocompatible, allows for biomolecular attachment, such as conjugation to antibodies and other targeting moieties for biological interactions. Syphilis, caused by the etiological agent, spirochetal bacterium Treponema pallidum (Tp), is one of the most common infectious venereal diseases.15, 16 The diagnosis of syphilis requires epidemiologic exposure and characteristic symptoms and signs, juxtaposed to laboratory tests, the mainstay of which remains serology. Current routine screening for syphilis begins with a non-specific text – rapid plasma reagin (RPR), the result of which, if positive, is confirmed by a specific treponemal test, such as chemiluminescence microparticle immunoassay (CMIA), enzyme-linked immunosorbent assays (ELISA), treponema pallidum particle agglutination assay 2


Page 2 of 24

Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


16

(TPPA), and toluidine red unheated serum test (TRUST).

Sufficient, well-trained health

professionals and laboratory resources become a necessity for conventional testing, which means a tremendous effort and high costs. Despite alternative means, such as the lateral-flow immunoassay (LFIA) based colloid gold nanoparticles with limitation of sensitivity remains a major obstacle to expanding their use as robust and reliable medical instruments for infectious disease screening. To outsmart the aforementioned current techniques, we elaborately designed a novel LFIA system mediated by modified nanoparticles-a poly (acrylic acid) (PAA) coat that presents the GMNs with a

hydrophilic

surface

compatible

for

biological

interactions.

With

a

linker,

1-ethyl-3-(3-diethyllaminopropyl) carbodiimide (EDC), targeted moieties can be efficiently conjugated to the carboxylic groups of PAA coated GMNs (PGMNs). Therefore, the PGMNs-mediated conjugates may be conceived as a colloidal, mono-dispersed particle probe to accurately detect target with highly sensitivity and specifity based on LFIA strips in clinical diagnostics. ■EXPERIMENTAL SECTION Materials and reagents. Fe3O4/Au/Fe3O4 composite nanoparticle (5 mg/mL) with nanoflower structure was gifts from Xi’an GoldMag Nanobiotech Co., Ltd. (Xi’an, PRC). The LFIA strips using PGMNs as particle probes were produced by Xi’an GoldMag Nanobiotech Co., Ltd. Human sera used in the laboratory test were provided by Shaanxi Provincial People’s Hospital (Xi’an, PRC). Buffer was prepared according to standard laboratory procedures. Water (18.2 MΩ· cm) purified by Barnstead Nanopure system was used for all the work in this report. All chemicals listed below were of analytical grade and purchased from reputable vendors. Cetyltrimethylammonium bromide (CTAB, 99% purity), 1-ethyl-3-(3-diethyl-laminopropyl) carbodiimide (EDC) and poly-acrylic acid (PAA sodium salt: Mw=2000, 8000 and 15000) were from Sigma-Aldrich (St. Louis, USA). Recombinant Treponema pallidum antigen (r-Tp) was obtained from Xiamen University (Xiamen, PRC) without further purification. Goat anti-rabbit IgG was purchased from Joey Bioscience Inc. (Shanghai, PRC). Reference samples, including anti-Tp (1, 2, 4 and 8 NCU/mL), anti-HIV-1 (8 NCU/mL), anti-HCV (8 NCU/mL) and anti-HBsV (8 NCU/mL) antibodies (NCU/mL refers to National Health Laboratory Center Units, China) were from Beijing Controls & Standards Co., Ltd. (Beijing, PRC). Negative control was the sera

from Shaanxi Provincial People’s Hospital and have been confirmed that there is no 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

anti-Tp in sera. Sera of 1,020 for syphilis screening were from Shaanxi Provincial People’s Hospital (340 cases), First Affiliated Hospital of Medical College of Xi’an Jiaotong University (340 cases) and Xiangya Hospital Central-South University (340 cases), respectively. Commercial LFIA strips were obtained from the Treponema pallidum antibodies colloid gold diagnostic kit, ACON Biotech Co., Ltd. (Hangzhou, PRC). Apparatus. A Hitachi H-600 Transmission Electron Microscope (TEM) was employed to acquire images of particles for structural assessment whereas size (diameter) and zeta potential was characterized in a Malvern Zetasizer ZS Instrument. A Shimadzu 2550 UV-Visible scanning spectrophotometer was used to determine the surface plasmon resonance (SPR). PAA coated particles were washed 3 times followed by 8 h of drying at 90°C, and then was assessed by a Bruker Vector 22 Fourier Transform-Infrared Spectroscopy (FT-IR) and a Metter SDTA 851e Thermo Gravimetric Analyzer (TGA). Surface modified GMNs with PAA. A pure core/shell of Fe3O4/Au was obtained by dispersing the GoldMag nanoflower (Fe3O4/Au/Fe3O4) with the cationic surfactant, CTAB.1, 17 Ten mL of CTAB (5 mmol/L) was mixed with 10 mL of nanoflower (approximately 5 mg/mL), stirred gently for 1 h and particles magnetically purified. A PAA solution was prepared by adding 1.88 g of PAA to 100 mL of deionized water. One mL of CTAB-capped GMNs particle solution was added to 2 mL of PAA solution and mixed on a shaker for 60 min. PGMNs were magnetically purified and suspended in 1 mL of deionized water. Conjugation of PGMNs with targeted moieties. EDC chemistry was used to covalently bond targeted moieties to PAA coated particles; 1 mg of particles was equilibrated in phosphate buffer (1×PB, pH 7.2) containing 20 µL of EDC (5 mg/mL). Then, 100 µg of r-Tp antigens or goat-anti rabbit IgG（1 mg/mL）were added to the EDC activated particles for incubating 1 h (Lowry protein concentration assay were explored to determine the conjugation efficiency by using bovine serum albumin (BSA) as a protein standard). The conjugation of PGMNs and protein was separated by a magnetic field and the uncoupled components were removed from the system. Blocking buffer (1×PB buffer, pH 7.2, containing 2.5% BSA and 1.25% calf serum) was added to the conjugates and the mixture was incubated for another 1 h; after 2 h of incubation, r-Tp functionalized particles or goat anti-rabbit IgG-PGMNs conjugates, were magnetically purified and then suspended in PB suspension buffer (1×PB, pH 7.2, containing 1% BSA) at 4°C until use. 4


Page 4 of 24

Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Detection of anti-Tp antibodies with r-Tp particle probes. Using a BioJetTM (HM3010, BioDot Inc., USA), capture Tp antigen (c-Tp) and rabbit IgG were pre-immobilized in a defined test line (T-line) and a control line (C-line) on a porous nitrocellulose membrane (NC), respectively. And then probe solution containing PGMNs-rTp antigens and PGMNs-goat anti rabbit IgG at a ratio of 5:1 was dispensed on the conjugate pad of LFIA strips (These strips were stored in a sealed aluminum foil bag with desiccant silica gel at room temperature and remained stable for 18 months). This was followed by pipetting 70 µL of human sera on to the layered strip – experiments included both negative and positive sera. Serial Treponema pallidum-positive sera with dilutions of 1:2, 1:20, 1:100, 1:200, 1:1000 and 1:2000 were firstly used to test the system. Then the clinical reference samples ranging from 1 NCU/mL to 8 NCU/mL were tested to determine detection limitation of the assay. For this assay, coloration will yield by forming a sandwich structure (c-Tp antigen/Tp antibody/PGMNs-rTp conjugates) and the intensity is related with the amount of captured particle conjugates (T-line). The simultaneous presence of a red band assures that there is specific binding of goat anti-rabbit IgG-particle conjugates to immobilized rabbit IgG (C-line). Assay specificity was assessed with the reference reagents, including anti-Tp antibodies (4 NCU/mL), anti-HBVs (8 NCU/mL), anti-HCV (8 NCU/mL) and anti-HIV-1 (8 NCU/mL). Contrast experiments analysis was also applied in LFIA upon application of GMNs to compare the strengths and weakness of PGMNs. Clinical trials for Syphilis screen with PGMNs-rTp LFIA strips. Clinical tests for 1,020 of human sera were performed in three hospitals, involving First Affiliated Hospital of Medical College of Xi’an Jiaotong University (Xi’an, PRC), Xiangya Hospital Central-South University (Changsha, PRC) and Shaanxi Provincial People’s Hospital (Xi’an, PRC). Each sample was tested and analyzed by our PGMNs-rTp LFIA strips and the commercial strips as comparison. Irreconcilable diagnosis in respective hospital was addressed via third party tests that include TPPA (Fuji, Japan) in First Affiliated Hospital of Medical College of Xi’an Jiaotong University, CMIA (Architect I2000) in Xiangya Hospital Central-South University and TRUST in Shaanxi Provincial People’s Hospital. All diagnostic procedures were performed according to manufacturers’ instructions. Based on the 2×2 contingency table, the positive, negative and total agreements were calculated to evaluate the accuracy of our method. Furthermore, the statistics methods, McNemar 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

chi-square test and kappa test, were applied to determine whether the positive rates are the same and measure of association between two methods respectively. ■RESULTS AND DISCUSSIONS Coating of GMNs with PAA. Synthesis and characterization of gold magnetic nanoparticles (nanoflower structure) were described previously.17 The molecule structure of CTAB and PAA, as well as general scheme to functionalize nanoflower GMNs with CTAB and PAA coating, is shown in Figure 1. The addition of cationic surfactant CTAB to the Fe3O4/Au/Fe3O4 particles inhibits the absorption of petals to the Fe3O4/Au core shell structure; thus TEM image in Figure 1 (middle) showed most of petals were removed and pure Fe3O4/Au core/shell structures were attained. Furthermore, CTAB stabilized the core/shell structure by neutralizing layers of surface positive charge to prevent aggregation. The CTAB-capped particles displayed an average diameter of 32 nm and were stable in their original high-CTAB containing solution for several months. Taking into consideration the positively charged surface and average size of CTAB-capped GMNs particle, PAAs with different molecule weight were chosen to modify the particles. Red and clear solutions were observed respectively when PAAs with Mw of 8,000 and 15,000 were added into the particle solution, while aggregations were formed when Mw of PAA was about 2,000. This result suggested that the length of polyelectrolyte may serve as an important parameter for the extent of polymerization in order to provide nanoparticles with complete surface coverage. avoid bridging flocculation and obtain adequate stability. Figure 1 shows TEM images of particles in the surface modified process step by step. PGMNs are well dispersed without iron oxide deposits on their surface, as evident in Figure 1 (right) and Figure 2A. In order to confirm that these particles have been successfully coated, PGMNs were analyzed by FT-IR and TGA.18, 19 As shown in Figure 2B, the spectrum of PGMNs shows the characteristic carboxylate bands from PAA while the distinctive peaks of CTAB are invisible in the PAA-Fe3O4/Au particle spectrum which provides evidence of the polymer on the particles. In further analysis, spectra for the CTAB-capped GMNs showed the distinctive absorption peaks centered at 2,920, 2,850, 1,401, and 960 cm-1, which represented symmetric (~2,920 and 2,850 cm-1) and asymmetric (1,401 cm-1) stretching of the C-H bond in CTAB, and quaternary amine (960 cm-1) stretch of CTAB, respectively.20, 21 In the spectra of PAA, the broad and strong band at 3,399 cm-1 is stretching of the O-H group. The peaks at 2,926 and 1,451 cm-1 are symmetric 6


Page 6 of 24

Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


stretching and scissoring bending vibrations of –CH2–. In neutral solutions, carboxyl groups on PAA deprotonate thus the bands at 1,550 and 1,400 cm-1 are characteristic of carboxylate stretch. Furthermore, these absorption bands suggested that polymers were modified to the surface of the particle via carboxyl groups of PAA interacting with trimethyl ammonium group of CTAB.22, 23 TGA analysis of GMNs and PGMNs showed that raw particles did not exhibit significant weight loss from 100 to 900°C, while weight of modified particles PGMNs were in a gradual decline, which could be attributed to dehydration and combustion of the organic molecules attached to GMNs (Figure 3C). Herein, the amount of the PAA attached to the surface of particle was about 45%. Stability of PGMNs. The surface charge of these particles could be altered depending on its environmental pH and ionic strength. Since pH is a more crucial parameter in determining the surface charge, we determined the zeta potential of these particles in neutral pH (water) to test whether the PAA coat had any effect on the surface potential. As shown in Figure 2D, CTAB capped GMNs were positively charged (+25.97 mV) due to the positive charge of the trimethyl ammonium group –N (CH3)3+ of CTAB, but became negatively charged (-58.90 mV) after PAA coating on NPs surface. This reversal of zeta potential upon PAA coating indicated the presence of PAA on the surface24, 25 – the pKa of PAA in absence of various salt reported by literature range from ~5.5 to 6.5.22, 23 When PGMNs were added into sodium chloride solution with an ionic strength (I) of 30 mmol/L or 1×PB buffer at pH 7.2, only a slight decrease in zeta potential of PGMNs (Figure 2D) was observed due to ion screen effects of PAA in electrolyte solution. The high value of zeta potential substantiates the purpose for selecting PAA to coat CTAB capped GMNs – keeping the particle mono-dispersed and colloidal via electrostatic repelling. Indeed, PGMNs thus maintained mono-dispersed and colloidal stability for ~6 months. The SPR band, an intense surface plasmon absorption band, occurs at a frequency of the electromagnetic waves that was identical to that of the collective oscillation of the conduction electrons in metal nanoparticles. Hence, qualitative measures for characterizing metallic nanoparticles, such as surface microenvironment, stability in aqueous solution, particle numbers, and inter-particle interactions (e.g. bridging), could be provided by the position and intensity of SPR bands.26, 27 In Figure 3A, the SPR band showed a blue-shift from 532 nm (CTAB capped particles) to 528 nm, and only a slight decrease in intensity was detected. The result suggested that 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

PGMNs were mono-dispersed and particle bridging was kept to a minimum level, considering that the intensity of SPR band was in accordance with the amount of individual particles dispersed in the same solution which has been studied extensively.28-30 We further tested the stability of PGMNs by subjecting them to an electrolyte solution (I=30 mmol/L) and PB buffer (1×, pH 7.2). Results showed (Figure 3B) that there was only a slight decrease of SPR intensity, and no red-shift of characteristic peak was observed, indicating the particle were in a good dispersion. Thus, aforementioned analysis showed that the stability of PGMNs was not affected by general ion screen, and such ionic independence stability potentially due to the PAA wrapping provides the particle steric stabilization to prevent the formation of aggregation and flocculation.31, 32 Layer-by-layer technique is a well-established method to build multilayer coating on flat solid surfaces or particle surface by using different building blocks of small molecules and/or polyelectrolytes, although coating of nanoparticles with macromolecules remains a tremendous challenge.30, 32 Problems associated with coating of polyelectrolyte around nanoparticles with a high curvature include 1) aggregation resulting from cross-linking of the particles by the polyelectrolyte chains, and 2) the separation of the unbound polyelectrolyte after fabrication process.25, 33-38 In addition, parameters, such as polymer chain length in order to provide particle enough stability, functional groups for downstream bioconjugation, and inter-forces between particle and polymer layer, should be identified. These challenges were potentially addressed with PAA, a polymer bearing plentiful carboxyl groups in its backbone and could provide enough electrical and steric stability as a polymer coat. Conjugation of PGMNs with targeted moieties. For immunoassay development, PGMNs particles were conjugated to r-Tp antigens to construct particle probes for detection of anti-Tp antibodies. Bioconjugation of particles with targeted moieties was confirmed by dynamic light scattering (DLS) measurement, SPR band analysis, TEM imaging and Lowry protein concentration assay. As shown in Figure 4A, the average diameter of conjugated particles increased from 64 nm to 85 nm. Considering 10 nm as an average hydrodynamic size of r-Tp antigen, the reasonable increase of hydrodynamic size after the conjugation suggested that the antigens were effectively coupled to the particle.30,

39

As shown in Figure 4B, UV-Vis

spectroscopy revealed a corresponding red-shift of SPR, from 528 nm to 533 nm due to r-Tp antigen conjugation, indicative of the increase of surface coating thickness (i.e. conjugated protein) 8


Page 8 of 24

Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and change of refractive index changes from a non-protein (PAA) to that of a protein.30, 40-42 TEM data showed that protein conjugated particles were in mono-dispersion and colloidal stability (Figure 4C). Similar procedure was performed for coupling goat anti-rabbit IgG with PGMNs, and the resultant conjugates were also in a colloidal stability and mono-dispersion. By Lowry protein determination and systematic studies, parameters involving proteins’ content and pH value of buffer were optimized – a maximum amount of 38 µg of antigens per mg of PGMN particles for r-Tp coupling, and 52 µg of antibodies/mg of PGMNs for goat anti-rabbit IgG conjugation. Detection of anti-Tp antibodies with PGMNs-rTp LFIA strip. Serological diagnosis of syphilis, coupled with enzyme immunoassays, gained its popularity 10 years ago, especially when portable LFIA recently became the integral part of such avenues.43,44 For a near quantitative, more accurate, and affordable diagnostic detection of syphilis, we elaborately applied our PGMN technique to the LFIA concept. Particles conjugated to r-Tp antigen were used as probes in LFIA to detect anti-Tp antibodies in sera of patients. Figure 5 shows a schematic diagram of detection principles for anti-Tp antibody and the apparatus employed for LFIA. Two types of conjugates, PGMNs-rTp and PGMNs-goat anti rabbit IgG, were dispensed onto the conjugate pad and dried there. Upon addition of human serum (containing analyte) onto the adjacent sample pad, the conjugates are rehydrated and consequently released into the migrating fluidic serum. If the testing serum happens to contain Tp antibodies, the PGMNs-rTp probes are able to recognize and link to the Tp antibodies. Driven by capillary force, the mixture of the fluids (i.e. two types of conjugates and potential Tp antibodies) then migrates across both the T-line and the C-line where c-Tp antigens and rabbit IgG are pre-immobilized, respectively. Subsequently, agglutination of conjugates released from conjugate pad, such as the triplet constructs of PGMNs-rTp antigens, anti-Tp antibodies, and c-Tp antigens in a sandwich format, will form a red coloration in the “T-line” on the NC membrane, where the color density is proportional to the amount of analyte in the sample.45, 46 In contrast, PGMNs-goat anti-rabbit IgG conjugates are recognized by rabbit IgG and colorate in the C-line, an internal control that reflects the effective release of particle-secondary antibody conjugates from the conjugate pad thereby. However, healthy sera without anti-Tp antibodies pass the T-line without coloration (Supplementary Figure S1). Figure 6A is representative of a typical LFIA result. A strong positive Tp antibody serum sample from Shaanxi Provincial Peoples’s Hospital was firstly used to test the strips. The serum 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

was analyzed in serial dilutions (1:2, 1:20, 1:100, 1:200, 1:1,000 and 1:2,000) for a comparative visualization of detection. The result showed that T-line could be visually recognized even at 2,000-fold dilution. 1 NCU/mL, the minimum amount of target analyte that has reacted with the particle probes causing coloration in the T-line is considered as the detection limit of this assay (Figure 6B). Herein, 1 NCU/mL refers to the minimum content of anti-Tp in a specific serum sample that can be checked out by using reference standard diagnostic kit. All experiments were conducted with a negative control to ensure that no non-specific interactions occurred between the migrating PGMNs-rTp probes and T-line bound c-Tp antigens. In addition, specificity of the assay for Tp antibodies was tested against antibodies of other pathogens, such as anti-HBsV, anti-HCV and anti-HIV-1 antibodies. Results showed that no coloration occurred in the T-line, suggesting that no nonspecific binding existed and thus the assay was indeed specific (Figure 6C). To compare the strengths or weaknesses of the above surface modification procedure, the similar analysis was also applied in LFIA strips using raw GMNs-rTp antigen conjugates (i.e. without PAA modification). As shown in Figure 6D, there was a visible gray line showed up when negative serum was used in detection, revealing the appearance of false-positive signals. Furthermore, presence of black smear-like aggregation in conjugate pad suggested a less effective release and migration of the particle probes based on GMNs that possessed a less colloidal stability compared with PGMNs. Thus, polyelectrolyte coating can introduce reactive carboxylic moieties not only for chemical absorption of protein to gold magnetic particle surface, but provide a more steric, ion independent stability and hydrophilic surface layers for downstream applications. We followed up with detecting Tp antibodies in patient sera with PGMNs-rTp LFIA strips. These immunoassays miniaturize the whole analytical chain from sampling to the detection of the analysts, and shed new light on the development of ideal POCT diagnosis devices based PGMNs. Clinical trials for syphilis screen using PGMNs-rTp LFIA strips. Due to lack of symptoms or lesions among participants who were all apparently healthy, syphilis diagnosis was based only on serologic testing.47 Samples tested positive to PGMNs-rTp LFIA strips and commercial strips were considered as serologically active syphilis, whilst those which were seronegative to both strips were accordingly uninfected. The defined agreement of seropositive and seronegative results for syphilis serology tests using PGMNs-rTp LFIA strips in comparison with commercial strips reached 97.7% and 97.1%, respectively (Table 1). In regard to the use of PGMNs-rTp LFIA strips, 10


Page 10 of 24

Page 11 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the total agreement covered 993 out of 1,020 result readings (97.4%) (Supplementary Table S1), which were higher than the 75% standard regulation from the State Food and Drug Administration (SFDA, China). Irreconcilable diagnosis, including 18 cases of PGMNs-rTp LIFA (+)/commercial LIFAs (-) as well as 9 case of PGMNs-rTp LIFA (-)/commercial LIFAs (+) were addressed via third party tests in respective hospitals (Table 1). In the case of First Affiliated Hospital of Medical College of Xi’an Jiaotong University, the TPPA gold standard test confirmed our 7 readings out of the 8 cases of the PGMNs-rTp LIFA(-)/commercial LIFAs (+) dispute. This result indicated PGMNs-rTp LFIA strips was able to serve as a more accurate testing comparing to the commercial counterpart. Among 8 cases of PGMNs-rTp LIFA (+)/commercial LIFAs (-) disagreement in Xiangya Hospital Central-South University, 5 cases were confirmed by CMIA, indicating PGMNs-rTp LIFA has higher sensitivity than the commercial counterpart. Thus, utilizing PGMNs as a carrier, higher sensitivity could be achieved compared to that of the conventional labeled with gold nanoparticles. As the method of primarily syphilis screening, the higher specificity and sensitivity suggest that our PGMNs-rTp LFIA strip is potentially a potent tool that can aid appropriate syphilis screening, minimizing any faulty omission. Therefore, due to their substantial advantages over the current diagnostic methodologies and commercially available products, such as the high sensitivity, better user friendly property, simplicity (e.g.no need of extra equipment for their use), stability at room temperature, and reasonable price, PGMNs-rTp LFIA strips could be recommended as a robust substitute for traditional diagnostic approaches, especially when a large amount of samples have to be analyzed. ■CONCLUSIONS A prerequisite for any potential bio-medical application for gold magnetic nanoparticles is proper functionalization which determines their biological interaction. For immunoassay development it is imperative that functionalization should maintain GMNs in a stable colloidal and mono-dispersed state with the ability to conjugate to targeted moieties. This was achieved with a simple and rapid coating procedure employing PAA in this study. We elaborately designed a PGMNs-mediated LFIA system in an attempt to outsmart the aforementioned current techniques. Upon conjugation with r-Tp, PGMNs-rTp probes were able to accurately detect anti-Tp antibodies in reference sample or sera with highly sensitivity and specificity based on LFIA strips. By 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

assessing the procedures for detections and diagnostic results of 1,020 sera from three independent hospitals, our novel PGMNs-rTp LFIA strips were proven easier and faster to perform, and of comparable reliability with the conventional tests, and shed new light on evidence-based practice for POCT. Finally, the data presented in this report is from the field clinical testing; therefore, it is possible to foresee that the conventional procedure for laboratory syphilis diagnosis might adapt to a new approach by using PGMNs-rTp LFIA strips for screening. ■ACKNOWLEDGEMENTS The author would like to acknowledge financial support from Key National Science and Technology Projects on “Key New Drug Creation and Manufacturing” (2012ZX09506001-001), Natural Science Foundation of Shaanxi Province (2012JM2003) and Special Scientific Research Project of Shaanxi Education Commission (12JK0628).

■ REFERENCES (1) Chao, X.; Guo, L.; Zhao, Y.; Hua, K.; Peng, M.; Chen, C.; Cui, Y.J. Drug Target 2011, 19, 161-170. (2) Jin, Y.; Jia, C.; Huang, S. W.; O’Donnell, M.; Gao, X. Nat. Commun. 2010, 6, 1-8. (3) Kamei, K.; Mukai, Y.; Kojima, H.; Yoshikawa, T.; Yoshikawa, M.; Kiyohara, G.; Yamamoto, T. A.; Yoshioka, Y.; Okada, N.; Seino, S.; Nakagawa, S. Biomaterials 2009, 30, 1809-1814. (4) Hwu, J. R.; Lin, Y. S.; Josephrajan, T.; Hsu, M.; Cheng, F.; Yeh, C.; Su, W.; Shieh, D. J. Am. Chem. Soc. 2008, 131, 66-68. (5) Okada, Y.; Takano, T. Y.; Kobayashi, N.; Hayashi, A.; Yonekura, M.; Nishiyama, Y.; Abe, T.; Yoshida, T.; Yamamoto, T. A.; Seino, S.; Doi, T. Bioconjugate Chem. 2011, 22, 887-893. (6) Zhao, M.; Zhang X.; Sen W.; Chen C.; Cui Y. J. Med. Colleges PLA 2009, 24, 239-243. (7) Park, H. Y.; Schadt, M. J.; Wang, L.; Lim, I. I.; Njoki, P. N.; Kim, S. H.; Jang, M. Y.; Luo, J.; Zhong, C. J. Langmuir 2007, 23, 9050-9056. (8) Wang, L.; Bai, J.; Li, Y.; Huang, Y. Angew. Chem. 2008, 47, 2439-2442. (9) Narayanan, S.; Sathy, B. N.; Mony, U.; Koyakutty, M.; Nair, S. V.; Menon, D. ACS Appl. Mater. Interfaces 2011, 1027-1035. (10) Larson, T. A.; Bankson, J.; Aaron, J.; Sokolov, K. Nanotechnology 2007, 18, 325101-325109. (11) Loaiza, O. A.; Jubete, E.; Ochoteco, E.; Cabanero, G.; Grande, H.; Rodriguez, J. Biosens. Bioelectron. 2010, 1-7. (12) Li, S.; Liu, H.; Liu, L.; Tian, L.; He, N. Anal. Biochem. 2010, 405, 141-143. (13) Gan, N.; Hou, J.; Hu, F.; Zheng, L.; Ni, M.; Cao, Y. Molecules 2010, 15, 5053-5065. (14) Cui, Y.; Hui, W.; Wang, H.; Wang, L.; Chen, C. Sci. China Ser. B 2004, 47, 152-158. (15) Lafond, R. E.; Lukehart, S. A., Clin. Microbiol. Rev. 2006, 19, 29-49. 12


Page 12 of 24

Page 13 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(16) Ma, K. F. Int. J. Lab. Med. 2012, 1994-1996. (17) Hui, W.; Shi, F.; Yan, K.; Peng, M.; Cheng, X.; Luo, Y.; Chen, X.; Roy, V. A. L.; Cui, Y.; Wang, Z. Nanoscale 2012, 4, 747-751. (18) Lin, C.; Lee, C.; Chiu W. J. Colloid Interface Sci. 2005, 291, 411-420. (19) Zhang, T.; Ge, J.; Hu, Y.; Yin, Y. Nano Lett. 2007, 7, 3203-3207. (20) Nikoobakht, B.; El-Sayed, M. A. Langmuir 2001, 17, 6368-6374. (21) Wijaya, A.; Hamad-Schifferli, K. Langmuir 2008, 24, 9966-9969. (22) Choi, J.; Rubner, M. F. Macromolecules 2004, 38, 116-124. (23) Kato, N.; Schuetz, P.; Fery, A.; Caruso, F. Macromolecules 2002, 35, 9780-9787. (24) Boyer, C.; Whittaker, M. R.; Chuah, K.; Liu, J.; Davis, T. P. Langmuir 2009, 26, 2721-2730. (25) Elbakry, A.; Zaky, A.; Liebl, R.; Rachel, R.; Goepferich, A.; Breunig, M. Nano Lett. 2009, 9, 2059-2064. (26) Yu, C.; Varghese, L.; Irudayaraj, J. Langmuir 2007, 23, 9114-9119 (27) Sardar, R.; Park, J.W.; Shumaker-Parry, J. S. Langmuir 2007, 23, 11883-11889. (28) Verma, A.; Srivastava, S.; Rotello, V. M. Chem. Mater. 2005, 17, 6317-6322. (29) Rechberger, W.; Leitner, H. A.; Krenn, J. R.; Lamprecht, B.; Aussenegg, F. R. Opt. Commun. 2003, 220, 137–141. (30) Yuan, W.; Li, C. M. Langmuir 2009, 25, 7578-7585. (31) Tan, S. J.; Jana, N. R.; Gao, S.; Patra, P. K.; Ying, J. Y. Chem. Mater. 2010, 22, 2239-2247. (32) Kozlovskaya, V.; Kharlampieva, E.; Khanal, B. P.; Manna, P.; Zubarev, E. R.; Tsukruk, V. V. Chem. Mater. 2008, 20, 7474-7485. (33) Schneider, G.; Decher, G. Langmuir, 2008, 24, 1778-1789. (34) Yan, Y.; Such, G. K.; Johnston, A. P. R.; Lomas, H.; Caruso, F. ACS Nano 2011, 5, 4252-4257. (35) Zhao, Z.; Yin, L.; Yuan, G.; Wang, L. Langmuir 2011, 28, 2704-2709. (36) Kim, K.; Lee, J. W.; Shin, D.; Kim, K. L.; Shin, K. S. J. Phys. Chem. C 2010, 114, 9917-9922. (37) Takahashi, H.; Niidome, T.; Kawano, T.; Yamada, S.; Niidome, Y. J. Nanopart. Res. 2008, 10, 221-228. (38) Jans, H.; Liu, X.; Austin, L.; Maes, G.; Huo, Q. Anal. Chem. 2009, 81, 9425-9432. (39) Liu, X.; Dai, Q.; Austin, L.; Coutts, J.; Knowles, G.; Zou, J.; Chen, H.; Huo, Q. J. Am. Chem. Soc. 2008, 130, 2780-2782. (40) Wang, C.; Irudayaraj, J. Small 2010, 6, 283-289. (41) Jiang, T.; Liu, R.; Huang, X.; Feng, H.; Teo, W.; Xing, B. Chem. Commun. 2009, 1972-1974. (42) Kaur, K.; Forrest, J. A. Langmuir 2012, 28, 2736-2744. (43) Egglestone, S.; Turner, A. J. Commun. Dis. Pub. Health 2000, 3, 158-162. (44) Chen, C.; Wu, J. Sensors 2012, 12, 11684-11696. (45) Liu, C.; Jia, Q.; Yang, C.; Qiao, R.; Jing, L.; Wang, L.; Xu, C.; Gao, M. Anal. Chem. 2011, 83, 6778-6784. (46) Gubala, V.; Harris, L. F.; Ricco, A. J.; Tan, M. X.; Williams, D. E. Anal. Chem. 2011, 84, 487-515. 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(47) Juárez-Figueroa, L.; Uribe-Salas, F.; García-Cisneros, S.; Olamendi-Portugal, M.; Conde-Glez, C. J. Diagn. Micr. Infec. Dis. 2007, 59, 123-124.

14


Page 14 of 24

Page 15 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Molecular structures of cationic surfactant CTAB, polyelectrolyte PAA and the schematic illustration of the “over-all” process of surface modification; capping of the particles with surfactant CTAB and a follow-up coating of PAA. TEM images of nanoflower structure (left), CTAB-capped (middle) and PAA-coated GMNs (right) shows the state of particles during the coating process. Figure 2. (A) TEM image of PGMNs are in a well diapered state and without iron oxide deposits on their surface. (B) FT-IR spectroscopy of CTAB-capped GMNs, pure PAA and PGMNs. (C) TGA analysis of raw GMNs and PGMNs. (D) Zeta potential of raw GMNs (nanoflower structure), CTAB-capped GMNs and PGMNs suspended in water, PGMNs suspended in high electrolyte solution (I=30 mmol/L) and PGMNs suspended in physiological buffer. Figure 3. (A) UV-Vis spectroscopy of raw GMNs, CTAB-capped GMNs and PGMNs suspended in water. The characteristic absorption peaks for raw GMNs are 545 nm and 528 nm, respectively. (B) UV-Vis spectroscopy of PGMNs suspended in water, electrolyte solution (I=30 mmol/L), and 1×PB buffer (pH 7.2) showed no significant change of SPR bands. Figure 4. (A) Size distribution of PGMNs and PGMNs-rTp conjugates monitored by DLS analyzer. Reasonable increase of hydrodynamic size, from 64 nm to 85 nm, indicates the successful conjugation. (B) The UV-Vis spectroscopy of PGMNs-rTp conjugates and PGMNs-goat anti-rabbit IgG conjugates revealed a corresponding red-shift of SPR after raw GMNs conjugated with antibodies. (C) TEM image of PGMNs-rTp conjugates showed that protein particles conjugates were in well dispersion and colloidal stability. Figure 5. (A) A schematic diagram of PGMNs’ surface modification and synthesis of PGMNs particle probes’ and (B) shows the principles for anti-Tp antibody detection and the apparatus employed for PGMNs-rTp LFIA. Figure 6. Tests of PGMNs-rTp LFIA strips. The negative result was indicated by a colorless line at the T-line, whereas a positive result presented as a red line on the nitrocellulose membrane. (A) The serum was analyzed in serial dilutions (e.g. 1:2, 1:20, 1:100, 1:200, 1:1000 and 1:2000) and the sample in a dilution of 1:2000 was visually detected. (B) 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The minimum amount of target analyte that has reacted with the particle probes causing coloration in the T-line is considered as the detection limit of the assay, and the limit of detection of PGMNs-rTp LFIA strips was as low as 1 NCU/mL. (C) The specificity testing of PGMNs-rTp LFIA strips. (D) The comparative experiment of LFIA strips employing raw GMNs-rTp antigens showed there were discernable false-positive signals and a less effective release of conjugates. Table 1. Syphilis serology results of clinical tests.

16


Page 16 of 24

Page 17 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Molecular structures of cationic surfactant CTAB, polyelectrolyte PAA and the schematic illustration of the “over-all” process of surface modification; capping of the particles with surfactant CTAB and a follow-up coating of PAA. TEM images of nanoflower structure (left), CTAB-capped (middle) and PAA-coated GMNs (right) shows the state of particles during the coating process.

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 24

A

B

C

D

Figure 2. (A) TEM image of PGMNs are in a well diapered state and without iron oxide deposits on their surface. (B) FT-IR spectroscopy of CTAB-capped GMNs, pure PAA and PGMNs. (C) TGA analysis of raw GMNs and PGMNs. (D) Zeta potential of raw GMNs (nanoflower structure), CTAB-capped GMNs and PGMNs suspended in water, PGMNs suspended in high electrolyte solution (I=30 mmol/L) and PGMNs suspended in physiological buffer.

18


Page 19 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

B

Figure 3. (A) UV-Vis spectroscopy of raw GMNs, CTAB-capped GMNs and PGMNs suspended in water. The absorption peaks for raw GMNs are about 545 nm and 528 nm, respectively. (B) UV-Vis spectroscopy of PGMNs suspended in water, electrolyte solution (I=30 mmol/L), and 1× PB buffer (pH 7.2) showed no significant change of SPR bands.

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

B

C Figure 4. (A) Size distribution of PGMNs and PGMNs-rTp conjugates monitored by DLS analyzer. Reasonable increase of hydrodynamic size, from 64 nm to 85 nm, indicates the successful conjugation. (B) The UV-Vis spectroscopy of PGMNs-rTp conjugates and PGMNs-goat anti-rabbit IgG conjugates revealed a corresponding red-shift of SPR after raw GMNs conjugated with antibodies. (C) TEM image of PGMNs-rTp conjugates showed that protein particles conjugates were in well dispersion and colloidal stability.

20


Page 20 of 24

Page 21 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A A

B

Figure 5. (A) A schematic diagram of PGMNs’ surface modification and synthesis of PGMNs particle probes’ and (B) shows the principles for anti-Tp antibody detection and the apparatus employed for PGMNs-rTp LFIA.

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

B

C

D

Figure 6. Tests of PGMNs-rTp LFIA strips. The negative result was indicated by a colorless line at the T-line, whereas a positive result presented as a red line on the nitrocellulose membrane. (A) The serum was analyzed in serial dilutions (e.g. 1:2, 1:20, 1:100, 1:200, 1:1000 and 1:2000) and the sample in a dilution of 1:2000 was visually detected. (B) The minimum amount of target analyte that has reacted with the particle conjugates causing coloration in the T-line is considered as the detection limit of the assay, and the limit of detection of PGMNs-rTp LFIA strips was as low as 1 NCU/mL. (C) The specificity testing of PGMNs-rTp LFIA strips. (D) The comparative experiment of LFIA strips employing raw GMNs-rTp antigens showed there were discernable false-positive signals and a less effective release of conjugates.

22


Page 22 of 24

Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Syphilis serology results of clinical tests. Commercial strips

+

-

+

99

1

-

8

232

Third party reagent Total

a

Hospital

Assessment strip

+

-

Total

TPPA 0

1

1

7

340

Hospital

b

Assessment strip

9

+

120

8

CMIA 0

-

0

212

0

3 5

340 c

Hospital

Assessment strip

+

166

9

-

1

164

8 TRUST 4

5

0

1

340 Total Assessment strip

+

385

18

-

9

608

10 Total 4 1

1,020 a

9 13 27

Hospital First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Hospitalb Xiangya Hospital Central-South University and Hospitalc Shaanxi Provincial People’s Hospital

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

107x67mm (150 x 150 DPI)


Page 24 of 24

Polyelectrolyte-Coated Gold Magnetic Nanoparticles for Immunoassay

Recommend Documents