Biosensors and Bioelectronics 58 (2014) 314–319
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Plasmonic ELISA for the ultrasensitive detection of Treponema pallidum Xin-Min Nie a, Rong Huang a, Cai-Xia Dong a, Li-Juan Tang b, Rong Gui a,n, Jian-Hui Jiang b,nn a
Clinical Laboratory Centre of the Third Xiangya Hospital, Central South University, Changsha 410013, PR China State Key Laboratory of Chemo/Bio-sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China
b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 January 2014 Received in revised form 2 March 2014 Accepted 3 March 2014 Available online 12 March 2014
In this report, we have developed a plasmonic ELISA strategy for the detection of syphilis. Plasmonic ELISA is an enzyme-linked immunoassay combined with enzyme-mediated surface plasmon resonance (SPR) of gold nanoparticles (AuNPs). Immune response of the Treponema pallidum (T. pallidum) antibodies triggers the acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine to produce abundant thiocholine. The positive charged thiol, in turn, alters the surface charge distribution the AuNPs and leads to the agglomeration of the AuNPs. The induced strong localized SPR effect of the agglomerate AuNPs can, thus, allow the quantitative assay of T. pallidum antibodies due to the remarkable color and absorption spectral response changes of the reaction system. The plasmonic ELISA exhibited a quasilinear response to the logarithmic T. pallidum antibody concentrations in the range of 1 pg/mL–10 ng/mL with a detection limit of 0.98 pg/mL. Such a low detection limit was 1000-fold improvements in sensitivity over a conventional ELISA. The results of plasmonic ELISA in syphilis assays of serum specimens from 60 patients agreed with those obtained using a conventional ELISA method. The plasmonic ELISA has characteristics (analyte specific, cost-effective, ease of automatic, low limit of detection) that provide potential for diagnosis and therapeutic monitoring of syphilis. & 2014 Elsevier B.V. All rights reserved.
Keywords: Syphilis Treponema pallidum Enzyme-linked immunosorbent assay Surface plasmon resonance Gold nanoparticles
1. Introduction Syphilis, a sexually transmitted infection of considerable public health importance, is on the increase again (Chakraborty and Luck, 2008). The health effects resulted by syphilis infection are overwhelming. The dissemination of Treponema pallidum (T. pallidum), the causative organisms of syphilis, begins at the early infection until the pathogens throughout the infector's body, including skin, bones, central nervous and cardiovascular systems (Tipple et al., 2011; Tantalo et al., 2005; Martin et al., 2009 ). For untreated infected women, T. pallidum may be transmitted from mother to fetus during pregnancy or at birth (Nahmias et al., 2011). The systemic syphilis incursion also increases the risk of other infections, such as human immunodeficiency virus (HIV) (Park et al., 2011a, 2011b). Hitherto, syphilis remains an important global health problem and continues to challenge clinicians in diagnosis and therapeutic monitoring. Syphilis diagnosis mainly relies on serologic assays (Jiang et al., 2013; Herremans et al., 2010; Wellinghausen and Dietenberger, 2011), n
Corresponding author. Tel./fax: þ 86 731 88618548. Corresponding author. Tel.: þ 86 731 88664085; fax: þ86 731 88821916. E-mail addresses: [email protected] (R. Gui), [email protected] (J.-H. Jiang). nn
http://dx.doi.org/10.1016/j.bios.2014.03.007 0956-5663/& 2014 Elsevier B.V. All rights reserved.
because the causative organisms cannot be cultured in vitro (Jantzen et al., 2012) and the infection is characterized by long periods of latency in excess of twenty years (Zoni et al., 2013). Serologic assays include two types, nontreponemal and treponemal tests (Lipinsky et al., 2012; Castro et al., 2013, 2010; Zhang et al., 2012).The early tests are nontreponemal ones using nontreponemal lipoidal antigens to react with the antibodies to T. pallidum, such as toluidine red unheated serum test (TRUST) (Zhuang et al., 2012) and rapid plasma reagin test (RPR) (Sweene et al., 2013). Advantages of these tests are that they are inexpensive and simple to perform. However, the results of nontreponemal tests must be further confirmed by treponemal tests because of their low specificity and limited sensitivity (Seña et al., 2010). Overall, treponemal tests using T. Pallidum antigens have higher sensitivity and specificity than nontreponemal ones (Seña et al., 2010). Of all the treponemal tests cleared by the U.S. Food and Drug Administration (FDA) for diagnostic, confirmatory, and blood donor screening test purposes (Kania, et al. 2009), enzyme-linked immunosorbent assay (ELISA) is one of the most sensitive and specific techniques (Seña et al., 2010; Cheow et al., 2010). Enzyme-catalyzed signal transformation and amplification greatly promote the analytical performance. ELISA combined with recombinant T. pallidum antigens shows sensitivities of 94.7–99.1% and specificities more than 99% in clinical syphilis diagnosis (Seña et al., 2010; Cheow et al., 2010). It is also cost-effective and ease of automatic compared with other
X.-M. Nie et al. / Biosensors and Bioelectronics 58 (2014) 314–319
treponemal tests, such as treponema pallidum gelatin particle agglutination (TPPA) (Maple et al., 2010) and fluorescent treponemal antibody absorption assay (FTA-ABS) (Park et al., 2011a, 2011b). The problem of current ELISA strategies is that the detection limit of 1 ng/mL (Engvall and Perlmann, 1979; Lems-Van Kan et al., 1983; Fortin et al., 2009) is relatively high. It means these methods may be incapable if the concentrations of T. pallidum antibodies in serum specimens are quite low (Seña et al., 2010), for example, early primary infections. Yet, early diagnosis is of considerable importance in disease treatment and preventing the transmission of infection. In this context, sensitive and reliable detection of T. pallidum is of paramount importance for clinic diagnostics and therapeutics. Plasmonic ELISA has emerged as an ultrasensitive strategy enabling the detection of a few molecules of analytes with the naked eye (de la Rica and Stevens, 2012). Rather than the most common ELISA catalyzing a color change reaction of organic molecules, plasmonic ELISA performs an enzyme-mediated localized surface plasmon resonance (LSPR) (Wang et al., 2010) of metallic nanoparticles. LSPR is a special optical phenomenon conferred by the interaction of light with electrons on the metallic nanoparticle surfaces (Guo and Kim, 2012). The light, under which LSPR occurs, is strongly dependent on the metallic nanoparticle size, shape, surface and agglomeration state (Wang et al., 2010; Ogiso et al., 2013). Minor changes of the state of metallic nanoparticles will lead to immense optical property changes of them. In plasmonic ELISA, such LSPR can be quantitatively controlled and amplified by enzyme-mediated surrounding alteration of metallic nanoparticles (de la Rica and Stevens, 2012; Liu et al., 2013). It, thus, offers a potential approach for ultrasensitive assays of target molecules. We reported the development of an ultrasensitive plasmonic ELISA strategy based on an enzyme-linked immunoassay format with enzyme-mediated SPR of gold nanoparticles (AuNPs) for the detection of total antibodies to T. pallidum. The performance of plasmonic syphilis ELISA strategy was demonstrated using the serum specimens from sixty patients and compared to commercial TP-ELISA.
2. Materials and methods 2.1. Materials and reagents Acetylcholinesterase (AChE) (Type VI-S, 200–1000 U/mg), acetylthiocholine (ATC), streptavidin (SA) and (þ)-Biotin N-hydroxysuccinimide ester (BNHS) were obtained from Sigma Aldrich Chemical Co. Biotinylated mouse anti-human IgG was purchased from Jackson Immuno Research Laboratories Inc. The ELISA kits of T. pallidum antibodies for immunoassay and the 96-well polystyrene plates were purchased from Zhuhai Livzon Diagnostic Inc. When obtained, the 96-well polystyrene plates have been treated using 1 μg/mL T. pallidum antigens (TpN15, TpN17 and TpN47) in PBS buffer (10 mM, pH 7.4) at 4 1C over night and the wells were blocked with 5% fetal bovine serum for 1 h followed by washed three times using wash buffer. All other chemicals were of analytical grade and obtained from Beijing Dingguo Changsheng Biotechnology Co. Ltd. All solutions were prepared using ultrapure water, which was obtained through a Millipore Milli-Q water purification system. Peripheral blood samples from 60 patients were collected from the Third Xiangya Hospital via venipuncture in tubes without anticoagulant. After centrifuged at 3000 rpm for 5 min, the plasma was stored at 20 1C and thawed immediately before analysis. These 60 patients were 34 male and 26 female, aged between 20 and 70 years old. 2.2. Preparation of biotinylated AChE and citrate-stabilized AuNPs Biotinylated AChE was prepared using NHS-activated biotins to react efficiently with amino groups (–NH2) of AChE in PBS buffer.
315
Briefly, freshly prepared 150 μL of 10 mM NHS-biotin in dimethylformamide (DMF) was mixed with 2 mg AChE in 1 mL of 10 mM phosphate buffered saline (PBS, pH 7.4), and incubated for 2 h at room temperature. The solution was then desalted by Microspin G-50 columns (GE Healthcare, UK) to remove excess NHS-biotin. AuNPs were synthesized by citrate reduction of HAuCl4 according to reported protocols (Liu and Lu, 2006; Hill and Mirkin, 2006): 10 mL 38.8 mM trisodium citrate was rapidly added to a stirred boiling solution of HAuCl4 (100 mL, 1 mM), of which the color changed from pale yellow to deep red within several minutes. Then, the solution was heated under reflux for another 30 min to ensure complete reduction followed by slow cooling to room temperature. The average size of AuNPs was 13 72 nm as calculated from the transmission electron microscopy (TEM) image, with a concentration of 13 nM determined based on an extinction coefficient of 2.7 108 M 1 cm 1 at 520 nm for 13 nm AuNPs (Hill and Mirkin, 2006). The final AuNPs solution was stored at 4 1C for future use. 2.3. Analytical protocol of plasmonic TP-ELISA Commercial 96-well polystyrene microtiter plates modified with T. pallidum antigens were directly used. In typical assays, 100 μL of serum samples containing certain concentration of T. pallidum antibodies were added on the wells of the microtiter plates and incubated for 1 h. Then, the left liquid in the wells were poured out and biotinylated anti-human IgG (100 μL, 1.5 μg/mL) was added, for 1 h. After pouring out the liquid, streptavidin (SA) (100 μL, 4 μg/mL) was added and incubated for 30 min followed by adding biotinylated AChE (100 μL, 20 μg/mL) and incubating for another 30 min. Then, the plates were carefully washed five times with wash buffer and twice with distilled water. AChE-catalyzed hydrolysis reaction was performed by adding freshly prepared ATC (100 μL, 20 μM in hepes buffer) to each well and incubating 15 min at 37 1C. After that, 100 μL 1.6 nM citrate-stabilized AuNPs were added to each well. We measured the surface plasmon absorption spectra of AuNPs in the wavelength range from 400 nm to 800 nm in a 50 μL quartz cuvette on a UV-2450 UV–vis absorption spectrophotometer (Shimadzu, Japan) at room temperature. Dynamic light scattering analysis was performed using a Zetasizer 3000 HS particle size analyzer (Malvern Instruments, U.K.) to determine the hydrodynamic sizes of the AuNPs. Zeta potential (ζ) analysis was performed on a Zeta Sizer Nano ZS (Malvern Zetasizer Nano ZS90) to determine the surface charge distributions of the AuNPs. The TEM images were obtained on a field-emission high-resolution 2100F TEM (JEOL, Japan) at an acceleration voltage of 200 kV. Clinical serum specimens from 60 donors were analyzed by following the above mentioned protocol, but the optical density (OD) of the reaction solutions at 520 nm was measured using a RT6100 Microplate Spectrophotometer (Rayto, U.S.A.). Conventional ELISA was also performed by strictly following the introduction of the kit, to demonstrate the reliability of plasmonic ELISA.
3. Results and discussion 3.1. Analytical principle of plasmonic TP-ELISA The plasmonic TP-ELISA was based on an enzyme-linked sandwiched immunoassay format with enzymatic LSPR control of AuNPs. The analytical principle is illustrated in Scheme 1. Instead of an enzymatic-catalyzed color change of organic molecule in conventional TP-ELISA, plasmonic TP-ELISA utilizes an enzymatic reaction to alter the surroundings of AuNPs and control the LSPR effect of AuNPs. First, like common ELISA for T. pallidum
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Scheme 1. Schematic diagram of analytical principle of the plasmonic ELISA for T. pallidum antibody assay.
assays, solid-phase enzyme-linked immunoreactions are performed to capture T. pallidum antibodies. Recombinantare T. pallidum antigens, TpN47, TpN17, and TpN15, are immobilized on a polystyrene microtiter plate, and capture the T. pallidum antibodies from serum specimen to the microplate surface. With the addition of biotinylated anti-human IgG, complex products of antigen–antibody–antibody are formed on the microplate surface. Then, in the presence of streptavidin, these complexes further bind with biotinylated AChE via the intense interaction of biotin– streptavidin. AChE is a serine protease with high efficiency in hydrolyzing acetylthiocholine to produce a sulfhydryl compound, thiocholine. The formed thiol, in turn, attacks and covalently attaches to the surface atoms of citrate-stabilized AuNPs, contributing some positive charges of the surface of the AuNPs. The alteration of the surface charge distributions of AuNPs furnishes the electrostatic interaction between the nanoparticles, and thus changes the agglomeration state of AuNPs to induce strong localized plasmonic coupling between AuNPs with remarkable optical property changes of the reaction solution due to dramatically enhanced electromagnetic fields near the particle surfaces. Because the LSPR of AuNPs is selectively controlled by T. pallidumspecific immunoreactions, the resulting absorption spectral response can then give an indicator for the concentration of T. pallidum antibodies. Fig. 1 depicts typical absorption spectral responses of the plasmonic TP-ELISA strategy in the assay of T. pallidum antibodies. Different serum specimens from T. pallidum negative or positive donors were analyzed, besides the specimens negative for T. pallidum antibodies but positive to hepatitis C virus (HCV) antibodies and toxoplasma gondii (TOX) antibodies. The prepared serum specimens (100 μL of each) were incubated at the same time with a T. pallidum-coated microtiter plate in different wells followed by biotinylated AChE-catalyzed hydrolysis of acetylthiocholine (AChE: 20 μg/mL; acetylthiocholine: 20 μM) in the presence of 1.5 μg/mL biotinylated anti-human IgG for a well. With the addition of 100 μL citrate-stabilized AuNPs ( 1.6 nM), the solution displayed typical optical properties for well-dispersed AuNPs, with a red color and a single surface plasmon absorption peak centered at 520 nm in the well where T. pallidum negative serum specimen was added (curve a). The same phenomenon was also obtained in the well where T. pallidum negative but HCV and
Fig. 1. Typical absorption spectral responses of plasmonic ELISA in assays of T. pallidum antibodies: (a) T. pallidum negative serum specimen, (b) T. pallidum negative but HCV and TOX positive serum specimen, (c) T. pallidum positive serum specimen, and (d) T. pallidum positive serum specimen with unbiotinylated AChE instead of biotinylated AChE. The inset is the photograph for the corresponding systems.
TOX positive serum specimen was added (curve b). These results implied the biomolecules in normal serum specimens and other T. pallidum non-specific antibodies had little effect on citratestabilized AuNPs. In contrast, in the well where T. pallidum positive serum specimen was added, the solution rapidly changed into blue with the absorption peak decreased by 75% and a distinct concomitant red shift from 520 nm to 700 nm (curve c), a typical LSPR behavior of the AuNP agglomerations. Moreover, a further experiment using unbiotinylated AChE instead of biotinylated AChE was performed. As anticipated, no obvious color and absorption spectral response change were observed (curve d) compared with the test of normal serum specimen using plasmonic TP-ELISA. It revealed AChE that cannot bind with the complex products of the immunoreactions and was washed away in subsequent steps had no apparent impact on the stability of AuNPs. That is, the agglomeration of AuNPs was specific to the immobilized AChE on the microtiter plate. To further demonstrate the mechanism of plasmonic TP-ELISA, DLS, TEM and Zeta potential analysis were performed on the
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Fig. 2. Hydrodynamic sizes of AuNPs determined by DLS analysis (A) and Zeta potential analysis of AuNPs (B). Untreated and citrate-stabilized AuNPs, AuNPs in plasmonic ELISA in response to T. pallidum negative serum specimen and AuNPs in plasmonic ELISA in response to T. pallidum positive serum specimen.
AuNPs that obtained from experiments described in Fig. 1. Because the AuNP agglomeration usually displayed a large hydrodynamic diameter, DLS analysis could provide straightforward evidences for the agglomeration state of AuNPs. For the T. pallidum negative serum specimen resulted AuNPs, the average hydrodynamic diameter was 22 nm which was consistent with the size of untreated and well-dispersed AuNPs (Fig. 2A). While the diameter of the T. pallidum positive serum specimen resulted AuNPs was 187 nm, exhibiting a large agglomeration of AuNPs. Fig. 3 shows the states of AuNPs before or after T. pallidum antibody triggered plasmonic ELISA. It can be found that the citrate-stabilized AuNPs were well dispersed with an average diameter of 13 nm (Fig. 3A). In T. pallidum positive serum where plasmonic ELISA could be triggered, large aggregates of the AuNPs were observed (Fig. 3B), implying the surface charge changes of AuNPs due to AChE-catalyzed generation of thiocholine following immunoreactions. Zeta potential measurements of the AuNPs from different tests also suggested a substantial increase of surface positive charges in the reaction buffer, indicators of the formation of AuNP agglomeration (Fig. 2B). These findings were highly consistent with those obtained with the absorption spectral measurements
and further confirmed that the agglomeration state of AuNPs was specific to the enzyme-linked immunoreactions in plasmonic TP-ELISA. 3.2. Assay performance Fig. 4 displays typical absorption spectral responses of plasmonic TP-ELISA to T. pallidum antibodies of varying concentrations in serum. The final concentrations of T. pallidum antibodies were 0, 10 12, 10 11, 10 10, 10 9, and 10 8 g/mL. With the increasing concentrations of T. pallidum antibodies, the red color of the citrate-stabilized AuNPs gradually turned into purple or blue (Fig. 4B), suggesting the well-dispersed AuNPs were assembled into larger aggregates in the presence of higher concentrations of T. pallidum antibodies. Such visual observations were consistent with the absorption spectral measurements. The absorption peaks at 520 nm were found regularly decrease with increasing concentration of T. pallidum antibodies, while the absorption peaks at 700 nm were significant enhanced for concentrations more than 1 ng/mL. A plot of the decreased value of corresponding peak absorbance readings versus the concentrations of T. pallidum
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Fig. 3. TEM images of AuNPs. (A) Untreated and citrate-stabilized AuNPs; (B) AuNPs in plasmonic ELISA in response to T. pallidum positive serum specimen.
Fig. 4. Quantitative performance of conventional ELISA (A) and plasmonic ELISA (B): photographs and typical absorption spectral responses of the corresponding method in response to T. pallidum antibodies of varying concentrations, and the decreased value of corresponding peak absorbance readings versus T. pallidum antibody concentrations. Error bars are standard deviation of three repetitive experiments.
antibodies revealed a dynamic correlation between the peak absorbance and the antibodies concentration in the range from 0 to 10 8 g/mL. A quasilinear correlation was obtained to the logarithmic concentration ranging from 10 12 g/mL to 10 8 g/mL with a detection limit of 0.98 pg/mL in terms of the rule of 3 times standard deviation over the blank response. Such a low detection limit and a wide dynamic range were much better (at least 1000fold improvement) than conventional ELISA for T. pallidum antibody assays (Fig. 4A). For conventional ELISA in which horseradish peroxidase (HRP) was used to catalyze a color change of an organic compound, no significant signals were obtained when the concentrations of T. pallidum antibodies were lower than 10 9 g/mL (Fig. 4A). Accordingly, the color change of a sample observed using naked eye can only be achieved when the concentration of
T. pallidum antibodies above 10 9 g/mL. By contrast, using naked eye, a color change can be observed in the sample with a concentration of T. pallidum antibodies as low as 10 11 g/mL in plasmonic TP-ELISA, 100-fold improvement compared with conventional ELISA. Furthermore, the plasmonic TP-ELISA with a colorimetric format was found to show very desirable reproducibility. The relative standard deviations (RSDs) of peak absorbance readings at 520 nm were 1.1%, 5.3%, 4.3%, 4.7%, and 3.5% in three repetitive assays of 10 8 g/mL, 10 9 g/mL, 10 10 g/mL, 10 11 g/mL, and 10 12 g/mL, respectively. Therefore, we might conclude that the developed plasmonic ELISA held potential for quantitative assay of T. pallidum with desirable sensitivity and reproducibility. Furthermore, 60 human serum specimens from patients were analyzed using the proposed plasmonic TP-ELISA and compared
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81101828, and 21205034), and Fundamental Research Funds for the Central Universities (No. 2011JQ030).
References
Fig. 5. The results of T. pallidum antibody assays of 60 serum specimens from different donors: plasmonic ELISA versus conventional ELISA.
with a conventional ELISA. The results obtained by the proposed method were in good agreement with those obtained by other two methods (Fig. 5). These results suggest that the plasmonic TP-ELISA was comparable with clinical assays, and might hold promise as a viable technique for the determination of T. pallidum antibodies in serum samples. 4. Conclusion In conclusion, we have developed a novel plasmonic ELISA for the detection of T. pallidum. Immunoreaction triggered and enzyme mediated localized surface plasmon resonance between glod nanoparticles contributed to the strategy ultrasensitive and high specific in T. pallidum assays. Plasmonic ELISA not only reserved the advantages of the conventional ELISA, such as costeffective, ease of automatic and high specific, but also furnished greatly improved sensitivity. A detection limit of 1 pg/mL was achieved by plasmonic ELISA in T. pallidum assays with a colorimetric readout format. The assay results of serum specimens from 60 patients revealed the plasmonic TP-ELISA was applicable to the diagnosis and therapeutic monitoring of T. pallidum. Acknowledgments This work was supported by National Natural Science Foundation of China (NSFC) (Grant nos. 30300383, 21025521, 81072270,
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Plasmonic ELISA for the ultrasensitive detection of Treponema pallidum Xin-Min Nie a, Rong Huang a, Cai-Xia Dong a, Li-Juan Tang b, Rong Gui a,n, Jian-Hui Jiang b,nn a
Clinical Laboratory Centre of the Third Xiangya Hospital, Central South University, Changsha 410013, PR China State Key Laboratory of Chemo/Bio-sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China
b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 January 2014 Received in revised form 2 March 2014 Accepted 3 March 2014 Available online 12 March 2014
In this report, we have developed a plasmonic ELISA strategy for the detection of syphilis. Plasmonic ELISA is an enzyme-linked immunoassay combined with enzyme-mediated surface plasmon resonance (SPR) of gold nanoparticles (AuNPs). Immune response of the Treponema pallidum (T. pallidum) antibodies triggers the acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine to produce abundant thiocholine. The positive charged thiol, in turn, alters the surface charge distribution the AuNPs and leads to the agglomeration of the AuNPs. The induced strong localized SPR effect of the agglomerate AuNPs can, thus, allow the quantitative assay of T. pallidum antibodies due to the remarkable color and absorption spectral response changes of the reaction system. The plasmonic ELISA exhibited a quasilinear response to the logarithmic T. pallidum antibody concentrations in the range of 1 pg/mL–10 ng/mL with a detection limit of 0.98 pg/mL. Such a low detection limit was 1000-fold improvements in sensitivity over a conventional ELISA. The results of plasmonic ELISA in syphilis assays of serum specimens from 60 patients agreed with those obtained using a conventional ELISA method. The plasmonic ELISA has characteristics (analyte specific, cost-effective, ease of automatic, low limit of detection) that provide potential for diagnosis and therapeutic monitoring of syphilis. & 2014 Elsevier B.V. All rights reserved.
Keywords: Syphilis Treponema pallidum Enzyme-linked immunosorbent assay Surface plasmon resonance Gold nanoparticles
1. Introduction Syphilis, a sexually transmitted infection of considerable public health importance, is on the increase again (Chakraborty and Luck, 2008). The health effects resulted by syphilis infection are overwhelming. The dissemination of Treponema pallidum (T. pallidum), the causative organisms of syphilis, begins at the early infection until the pathogens throughout the infector's body, including skin, bones, central nervous and cardiovascular systems (Tipple et al., 2011; Tantalo et al., 2005; Martin et al., 2009 ). For untreated infected women, T. pallidum may be transmitted from mother to fetus during pregnancy or at birth (Nahmias et al., 2011). The systemic syphilis incursion also increases the risk of other infections, such as human immunodeficiency virus (HIV) (Park et al., 2011a, 2011b). Hitherto, syphilis remains an important global health problem and continues to challenge clinicians in diagnosis and therapeutic monitoring. Syphilis diagnosis mainly relies on serologic assays (Jiang et al., 2013; Herremans et al., 2010; Wellinghausen and Dietenberger, 2011), n
Corresponding author. Tel./fax: þ 86 731 88618548. Corresponding author. Tel.: þ 86 731 88664085; fax: þ86 731 88821916. E-mail addresses: [email protected] (R. Gui), [email protected] (J.-H. Jiang). nn
http://dx.doi.org/10.1016/j.bios.2014.03.007 0956-5663/& 2014 Elsevier B.V. All rights reserved.
because the causative organisms cannot be cultured in vitro (Jantzen et al., 2012) and the infection is characterized by long periods of latency in excess of twenty years (Zoni et al., 2013). Serologic assays include two types, nontreponemal and treponemal tests (Lipinsky et al., 2012; Castro et al., 2013, 2010; Zhang et al., 2012).The early tests are nontreponemal ones using nontreponemal lipoidal antigens to react with the antibodies to T. pallidum, such as toluidine red unheated serum test (TRUST) (Zhuang et al., 2012) and rapid plasma reagin test (RPR) (Sweene et al., 2013). Advantages of these tests are that they are inexpensive and simple to perform. However, the results of nontreponemal tests must be further confirmed by treponemal tests because of their low specificity and limited sensitivity (Seña et al., 2010). Overall, treponemal tests using T. Pallidum antigens have higher sensitivity and specificity than nontreponemal ones (Seña et al., 2010). Of all the treponemal tests cleared by the U.S. Food and Drug Administration (FDA) for diagnostic, confirmatory, and blood donor screening test purposes (Kania, et al. 2009), enzyme-linked immunosorbent assay (ELISA) is one of the most sensitive and specific techniques (Seña et al., 2010; Cheow et al., 2010). Enzyme-catalyzed signal transformation and amplification greatly promote the analytical performance. ELISA combined with recombinant T. pallidum antigens shows sensitivities of 94.7–99.1% and specificities more than 99% in clinical syphilis diagnosis (Seña et al., 2010; Cheow et al., 2010). It is also cost-effective and ease of automatic compared with other
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treponemal tests, such as treponema pallidum gelatin particle agglutination (TPPA) (Maple et al., 2010) and fluorescent treponemal antibody absorption assay (FTA-ABS) (Park et al., 2011a, 2011b). The problem of current ELISA strategies is that the detection limit of 1 ng/mL (Engvall and Perlmann, 1979; Lems-Van Kan et al., 1983; Fortin et al., 2009) is relatively high. It means these methods may be incapable if the concentrations of T. pallidum antibodies in serum specimens are quite low (Seña et al., 2010), for example, early primary infections. Yet, early diagnosis is of considerable importance in disease treatment and preventing the transmission of infection. In this context, sensitive and reliable detection of T. pallidum is of paramount importance for clinic diagnostics and therapeutics. Plasmonic ELISA has emerged as an ultrasensitive strategy enabling the detection of a few molecules of analytes with the naked eye (de la Rica and Stevens, 2012). Rather than the most common ELISA catalyzing a color change reaction of organic molecules, plasmonic ELISA performs an enzyme-mediated localized surface plasmon resonance (LSPR) (Wang et al., 2010) of metallic nanoparticles. LSPR is a special optical phenomenon conferred by the interaction of light with electrons on the metallic nanoparticle surfaces (Guo and Kim, 2012). The light, under which LSPR occurs, is strongly dependent on the metallic nanoparticle size, shape, surface and agglomeration state (Wang et al., 2010; Ogiso et al., 2013). Minor changes of the state of metallic nanoparticles will lead to immense optical property changes of them. In plasmonic ELISA, such LSPR can be quantitatively controlled and amplified by enzyme-mediated surrounding alteration of metallic nanoparticles (de la Rica and Stevens, 2012; Liu et al., 2013). It, thus, offers a potential approach for ultrasensitive assays of target molecules. We reported the development of an ultrasensitive plasmonic ELISA strategy based on an enzyme-linked immunoassay format with enzyme-mediated SPR of gold nanoparticles (AuNPs) for the detection of total antibodies to T. pallidum. The performance of plasmonic syphilis ELISA strategy was demonstrated using the serum specimens from sixty patients and compared to commercial TP-ELISA.
2. Materials and methods 2.1. Materials and reagents Acetylcholinesterase (AChE) (Type VI-S, 200–1000 U/mg), acetylthiocholine (ATC), streptavidin (SA) and (þ)-Biotin N-hydroxysuccinimide ester (BNHS) were obtained from Sigma Aldrich Chemical Co. Biotinylated mouse anti-human IgG was purchased from Jackson Immuno Research Laboratories Inc. The ELISA kits of T. pallidum antibodies for immunoassay and the 96-well polystyrene plates were purchased from Zhuhai Livzon Diagnostic Inc. When obtained, the 96-well polystyrene plates have been treated using 1 μg/mL T. pallidum antigens (TpN15, TpN17 and TpN47) in PBS buffer (10 mM, pH 7.4) at 4 1C over night and the wells were blocked with 5% fetal bovine serum for 1 h followed by washed three times using wash buffer. All other chemicals were of analytical grade and obtained from Beijing Dingguo Changsheng Biotechnology Co. Ltd. All solutions were prepared using ultrapure water, which was obtained through a Millipore Milli-Q water purification system. Peripheral blood samples from 60 patients were collected from the Third Xiangya Hospital via venipuncture in tubes without anticoagulant. After centrifuged at 3000 rpm for 5 min, the plasma was stored at 20 1C and thawed immediately before analysis. These 60 patients were 34 male and 26 female, aged between 20 and 70 years old. 2.2. Preparation of biotinylated AChE and citrate-stabilized AuNPs Biotinylated AChE was prepared using NHS-activated biotins to react efficiently with amino groups (–NH2) of AChE in PBS buffer.
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Briefly, freshly prepared 150 μL of 10 mM NHS-biotin in dimethylformamide (DMF) was mixed with 2 mg AChE in 1 mL of 10 mM phosphate buffered saline (PBS, pH 7.4), and incubated for 2 h at room temperature. The solution was then desalted by Microspin G-50 columns (GE Healthcare, UK) to remove excess NHS-biotin. AuNPs were synthesized by citrate reduction of HAuCl4 according to reported protocols (Liu and Lu, 2006; Hill and Mirkin, 2006): 10 mL 38.8 mM trisodium citrate was rapidly added to a stirred boiling solution of HAuCl4 (100 mL, 1 mM), of which the color changed from pale yellow to deep red within several minutes. Then, the solution was heated under reflux for another 30 min to ensure complete reduction followed by slow cooling to room temperature. The average size of AuNPs was 13 72 nm as calculated from the transmission electron microscopy (TEM) image, with a concentration of 13 nM determined based on an extinction coefficient of 2.7 108 M 1 cm 1 at 520 nm for 13 nm AuNPs (Hill and Mirkin, 2006). The final AuNPs solution was stored at 4 1C for future use. 2.3. Analytical protocol of plasmonic TP-ELISA Commercial 96-well polystyrene microtiter plates modified with T. pallidum antigens were directly used. In typical assays, 100 μL of serum samples containing certain concentration of T. pallidum antibodies were added on the wells of the microtiter plates and incubated for 1 h. Then, the left liquid in the wells were poured out and biotinylated anti-human IgG (100 μL, 1.5 μg/mL) was added, for 1 h. After pouring out the liquid, streptavidin (SA) (100 μL, 4 μg/mL) was added and incubated for 30 min followed by adding biotinylated AChE (100 μL, 20 μg/mL) and incubating for another 30 min. Then, the plates were carefully washed five times with wash buffer and twice with distilled water. AChE-catalyzed hydrolysis reaction was performed by adding freshly prepared ATC (100 μL, 20 μM in hepes buffer) to each well and incubating 15 min at 37 1C. After that, 100 μL 1.6 nM citrate-stabilized AuNPs were added to each well. We measured the surface plasmon absorption spectra of AuNPs in the wavelength range from 400 nm to 800 nm in a 50 μL quartz cuvette on a UV-2450 UV–vis absorption spectrophotometer (Shimadzu, Japan) at room temperature. Dynamic light scattering analysis was performed using a Zetasizer 3000 HS particle size analyzer (Malvern Instruments, U.K.) to determine the hydrodynamic sizes of the AuNPs. Zeta potential (ζ) analysis was performed on a Zeta Sizer Nano ZS (Malvern Zetasizer Nano ZS90) to determine the surface charge distributions of the AuNPs. The TEM images were obtained on a field-emission high-resolution 2100F TEM (JEOL, Japan) at an acceleration voltage of 200 kV. Clinical serum specimens from 60 donors were analyzed by following the above mentioned protocol, but the optical density (OD) of the reaction solutions at 520 nm was measured using a RT6100 Microplate Spectrophotometer (Rayto, U.S.A.). Conventional ELISA was also performed by strictly following the introduction of the kit, to demonstrate the reliability of plasmonic ELISA.
3. Results and discussion 3.1. Analytical principle of plasmonic TP-ELISA The plasmonic TP-ELISA was based on an enzyme-linked sandwiched immunoassay format with enzymatic LSPR control of AuNPs. The analytical principle is illustrated in Scheme 1. Instead of an enzymatic-catalyzed color change of organic molecule in conventional TP-ELISA, plasmonic TP-ELISA utilizes an enzymatic reaction to alter the surroundings of AuNPs and control the LSPR effect of AuNPs. First, like common ELISA for T. pallidum
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Scheme 1. Schematic diagram of analytical principle of the plasmonic ELISA for T. pallidum antibody assay.
assays, solid-phase enzyme-linked immunoreactions are performed to capture T. pallidum antibodies. Recombinantare T. pallidum antigens, TpN47, TpN17, and TpN15, are immobilized on a polystyrene microtiter plate, and capture the T. pallidum antibodies from serum specimen to the microplate surface. With the addition of biotinylated anti-human IgG, complex products of antigen–antibody–antibody are formed on the microplate surface. Then, in the presence of streptavidin, these complexes further bind with biotinylated AChE via the intense interaction of biotin– streptavidin. AChE is a serine protease with high efficiency in hydrolyzing acetylthiocholine to produce a sulfhydryl compound, thiocholine. The formed thiol, in turn, attacks and covalently attaches to the surface atoms of citrate-stabilized AuNPs, contributing some positive charges of the surface of the AuNPs. The alteration of the surface charge distributions of AuNPs furnishes the electrostatic interaction between the nanoparticles, and thus changes the agglomeration state of AuNPs to induce strong localized plasmonic coupling between AuNPs with remarkable optical property changes of the reaction solution due to dramatically enhanced electromagnetic fields near the particle surfaces. Because the LSPR of AuNPs is selectively controlled by T. pallidumspecific immunoreactions, the resulting absorption spectral response can then give an indicator for the concentration of T. pallidum antibodies. Fig. 1 depicts typical absorption spectral responses of the plasmonic TP-ELISA strategy in the assay of T. pallidum antibodies. Different serum specimens from T. pallidum negative or positive donors were analyzed, besides the specimens negative for T. pallidum antibodies but positive to hepatitis C virus (HCV) antibodies and toxoplasma gondii (TOX) antibodies. The prepared serum specimens (100 μL of each) were incubated at the same time with a T. pallidum-coated microtiter plate in different wells followed by biotinylated AChE-catalyzed hydrolysis of acetylthiocholine (AChE: 20 μg/mL; acetylthiocholine: 20 μM) in the presence of 1.5 μg/mL biotinylated anti-human IgG for a well. With the addition of 100 μL citrate-stabilized AuNPs ( 1.6 nM), the solution displayed typical optical properties for well-dispersed AuNPs, with a red color and a single surface plasmon absorption peak centered at 520 nm in the well where T. pallidum negative serum specimen was added (curve a). The same phenomenon was also obtained in the well where T. pallidum negative but HCV and
Fig. 1. Typical absorption spectral responses of plasmonic ELISA in assays of T. pallidum antibodies: (a) T. pallidum negative serum specimen, (b) T. pallidum negative but HCV and TOX positive serum specimen, (c) T. pallidum positive serum specimen, and (d) T. pallidum positive serum specimen with unbiotinylated AChE instead of biotinylated AChE. The inset is the photograph for the corresponding systems.
TOX positive serum specimen was added (curve b). These results implied the biomolecules in normal serum specimens and other T. pallidum non-specific antibodies had little effect on citratestabilized AuNPs. In contrast, in the well where T. pallidum positive serum specimen was added, the solution rapidly changed into blue with the absorption peak decreased by 75% and a distinct concomitant red shift from 520 nm to 700 nm (curve c), a typical LSPR behavior of the AuNP agglomerations. Moreover, a further experiment using unbiotinylated AChE instead of biotinylated AChE was performed. As anticipated, no obvious color and absorption spectral response change were observed (curve d) compared with the test of normal serum specimen using plasmonic TP-ELISA. It revealed AChE that cannot bind with the complex products of the immunoreactions and was washed away in subsequent steps had no apparent impact on the stability of AuNPs. That is, the agglomeration of AuNPs was specific to the immobilized AChE on the microtiter plate. To further demonstrate the mechanism of plasmonic TP-ELISA, DLS, TEM and Zeta potential analysis were performed on the
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Fig. 2. Hydrodynamic sizes of AuNPs determined by DLS analysis (A) and Zeta potential analysis of AuNPs (B). Untreated and citrate-stabilized AuNPs, AuNPs in plasmonic ELISA in response to T. pallidum negative serum specimen and AuNPs in plasmonic ELISA in response to T. pallidum positive serum specimen.
AuNPs that obtained from experiments described in Fig. 1. Because the AuNP agglomeration usually displayed a large hydrodynamic diameter, DLS analysis could provide straightforward evidences for the agglomeration state of AuNPs. For the T. pallidum negative serum specimen resulted AuNPs, the average hydrodynamic diameter was 22 nm which was consistent with the size of untreated and well-dispersed AuNPs (Fig. 2A). While the diameter of the T. pallidum positive serum specimen resulted AuNPs was 187 nm, exhibiting a large agglomeration of AuNPs. Fig. 3 shows the states of AuNPs before or after T. pallidum antibody triggered plasmonic ELISA. It can be found that the citrate-stabilized AuNPs were well dispersed with an average diameter of 13 nm (Fig. 3A). In T. pallidum positive serum where plasmonic ELISA could be triggered, large aggregates of the AuNPs were observed (Fig. 3B), implying the surface charge changes of AuNPs due to AChE-catalyzed generation of thiocholine following immunoreactions. Zeta potential measurements of the AuNPs from different tests also suggested a substantial increase of surface positive charges in the reaction buffer, indicators of the formation of AuNP agglomeration (Fig. 2B). These findings were highly consistent with those obtained with the absorption spectral measurements
and further confirmed that the agglomeration state of AuNPs was specific to the enzyme-linked immunoreactions in plasmonic TP-ELISA. 3.2. Assay performance Fig. 4 displays typical absorption spectral responses of plasmonic TP-ELISA to T. pallidum antibodies of varying concentrations in serum. The final concentrations of T. pallidum antibodies were 0, 10 12, 10 11, 10 10, 10 9, and 10 8 g/mL. With the increasing concentrations of T. pallidum antibodies, the red color of the citrate-stabilized AuNPs gradually turned into purple or blue (Fig. 4B), suggesting the well-dispersed AuNPs were assembled into larger aggregates in the presence of higher concentrations of T. pallidum antibodies. Such visual observations were consistent with the absorption spectral measurements. The absorption peaks at 520 nm were found regularly decrease with increasing concentration of T. pallidum antibodies, while the absorption peaks at 700 nm were significant enhanced for concentrations more than 1 ng/mL. A plot of the decreased value of corresponding peak absorbance readings versus the concentrations of T. pallidum
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Fig. 3. TEM images of AuNPs. (A) Untreated and citrate-stabilized AuNPs; (B) AuNPs in plasmonic ELISA in response to T. pallidum positive serum specimen.
Fig. 4. Quantitative performance of conventional ELISA (A) and plasmonic ELISA (B): photographs and typical absorption spectral responses of the corresponding method in response to T. pallidum antibodies of varying concentrations, and the decreased value of corresponding peak absorbance readings versus T. pallidum antibody concentrations. Error bars are standard deviation of three repetitive experiments.
antibodies revealed a dynamic correlation between the peak absorbance and the antibodies concentration in the range from 0 to 10 8 g/mL. A quasilinear correlation was obtained to the logarithmic concentration ranging from 10 12 g/mL to 10 8 g/mL with a detection limit of 0.98 pg/mL in terms of the rule of 3 times standard deviation over the blank response. Such a low detection limit and a wide dynamic range were much better (at least 1000fold improvement) than conventional ELISA for T. pallidum antibody assays (Fig. 4A). For conventional ELISA in which horseradish peroxidase (HRP) was used to catalyze a color change of an organic compound, no significant signals were obtained when the concentrations of T. pallidum antibodies were lower than 10 9 g/mL (Fig. 4A). Accordingly, the color change of a sample observed using naked eye can only be achieved when the concentration of
T. pallidum antibodies above 10 9 g/mL. By contrast, using naked eye, a color change can be observed in the sample with a concentration of T. pallidum antibodies as low as 10 11 g/mL in plasmonic TP-ELISA, 100-fold improvement compared with conventional ELISA. Furthermore, the plasmonic TP-ELISA with a colorimetric format was found to show very desirable reproducibility. The relative standard deviations (RSDs) of peak absorbance readings at 520 nm were 1.1%, 5.3%, 4.3%, 4.7%, and 3.5% in three repetitive assays of 10 8 g/mL, 10 9 g/mL, 10 10 g/mL, 10 11 g/mL, and 10 12 g/mL, respectively. Therefore, we might conclude that the developed plasmonic ELISA held potential for quantitative assay of T. pallidum with desirable sensitivity and reproducibility. Furthermore, 60 human serum specimens from patients were analyzed using the proposed plasmonic TP-ELISA and compared
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81101828, and 21205034), and Fundamental Research Funds for the Central Universities (No. 2011JQ030).
References
Fig. 5. The results of T. pallidum antibody assays of 60 serum specimens from different donors: plasmonic ELISA versus conventional ELISA.
with a conventional ELISA. The results obtained by the proposed method were in good agreement with those obtained by other two methods (Fig. 5). These results suggest that the plasmonic TP-ELISA was comparable with clinical assays, and might hold promise as a viable technique for the determination of T. pallidum antibodies in serum samples. 4. Conclusion In conclusion, we have developed a novel plasmonic ELISA for the detection of T. pallidum. Immunoreaction triggered and enzyme mediated localized surface plasmon resonance between glod nanoparticles contributed to the strategy ultrasensitive and high specific in T. pallidum assays. Plasmonic ELISA not only reserved the advantages of the conventional ELISA, such as costeffective, ease of automatic and high specific, but also furnished greatly improved sensitivity. A detection limit of 1 pg/mL was achieved by plasmonic ELISA in T. pallidum assays with a colorimetric readout format. The assay results of serum specimens from 60 patients revealed the plasmonic TP-ELISA was applicable to the diagnosis and therapeutic monitoring of T. pallidum. Acknowledgments This work was supported by National Natural Science Foundation of China (NSFC) (Grant nos. 30300383, 21025521, 81072270,
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