Plasmonic ELISA Research Articles

A silver auto-catalyzed plasmonic enzyme-linked immunosorbent assay for colorimetric and fluorescent detection of neutrophil gelatinase associated lipocalin (NGAL)

The conventional ELISA lacked sufficient sensitivity and accuracy for analyzing neutrophil gelatinase associated lipocalin (NGAL), a potential biomarker of ulcerative colitis. Here, we developed a dual-readout plasmonic ELISA with cascade amplification of alkaline phosphatase hydrolysis and O-phenylenediamine oxidation. In this novel plasmonic ELISA, alkaline phosphatase-conjugated antibodies capture NGAL from samples, alkaline phosphatase hydrolyzes 2-phospho-L-ascorbic acid into ascorbic acid, and then ascorbic acid blocks silver-catalyzed O-phenylenediamine oxidation by turning silver ions into silver microwires. Therefore, NGAL concentration is proportional to the decrease in chromogenic and fluorescent response. This novel plasmonic ELISA exhibited a linear range of 0.5–80 ng/mL, and the LOD was as low as 0.5 ng/mL. The sensitivity was superior to that of conventional ELISA and previous studies. The plasmonic ELISA exhibited favorable selectivity because of the specific antibody-antigen interaction and the robust cascade amplification. Besides, the dual-readout feature endowed the plasmonic ELISA with advantages in terms of convenience and compatibility. In summary, our study provided a novel and sensitive plasmonic ELISA for the diagnosis of ulcerative colitis.

Detection of biomolecules from nano to atto-gram through plasmonic Elisa via tuning the catalyst

A simple and affordable plasmonic ELISA platform is demonstrated using silver nanocrystals as chromogens, and nanocatalysts in the place of enzyme. Apart from ultralow detection limitup to 10 molecules of human Immunoglobulin G (2.5 × 10−18 g/1.68 × 10−19 M), we exhibit very broad dynamic range covering 11-log which goes up to 100 × 10−9 g/6.72 × 10−9 M. To achieve this broad dynamic range and highly controlled standard deviation, nanocatalysts were tuned in terms of shape, size, metallic ratio, surface modification and other parameters. A comparative study is also done between traditional chromogen to independently developed new silver chromogen, and among different catalysts. Our results indicate plasmonic ELISA with small size nanocatalysts may become a very powerful detection strategy for biomolecules.

Development of a Gold Nanoparticle-Based ELISA for Detection of PCV2.

In this new methodology, plasmonic ELISA (pELISA) was used to detect Circovirus porcine2 (PCV2) in serum samples without the need for plate reading equipment. This process occurs by adapting the conventional ELISA test with gold nanoparticles (AuNPs) to promote a color change on the plate and quickly identify this difference with the naked eye, generating a dark purple-gray hue when the samples are positive and red when the samples are negative. The technique demonstrated high efficiency in detecting samples with a viral load ≥ 5 log10 copies/mL. Plasmonic ELISA offers user-friendly, cost-effective, and reliable characteristics, making it a valuable tool for PCV2 diagnosis and potentially adaptable for other pathogen detection applications.

A label-free AuNPs-based ultrasensitive plasmonic ELISA by alkaline phosphatase and ferrous ion-initiated cascade amplification

Conventional ELISA methods have been widely used for disease diagnosis, yet the sensitivity and accuracy are still to be improved. Based on our label-free AuNPs-mediated plasmonic sensor, we developed an ultrasensitive plasmonic ELISA by integrating the plasmonic sensor and cascade amplification. In principle, alkaline phosphatase (ALP)-conjugated antibodies captured targets, and the ALP hydrolyzed 2-phospho-ascorbic acid (AA2P) into ascorbic acid (AA). Then, the released AA changed Fe3+ into Fe2+, which further triggered Fe2+-catalyzed H2O2-preventing aggregation of AuNPs by oxidizing Cysteine (FeHOAuC). Finally, the target content was negatively related with the AuNPs aggregation and the subsequent color variation. As a proof-of-concept, this novel plasmonic ELISA exhibited an ultralow LOD (0.1 ng/mL, around 4.4 pM) and a detection range (0.25–8.0 ng/mL) on detecting neutrophil gelatinase-associated lipocalin (NGAL) within 120 min. The sensitivity was superior to those of conventional ELISA and plasmonic ELISA. In summary, our study provided a novel and sensitive plasmonic ELISA for proteinic biomarker diagnosis.

A dichromatic plasmonic ELISA CD81 protein sensor for ultrasensitive detection of preeclampsia.

Preeclampsia (PE) seriously affects pregnant women and fetuses' health and causes maternal near-misses. CD81 has been confirmed to be a novel PE biomarker with great potential. Herein, a hypersensitive dichromatic biosensor based on the plasmonic enzyme-linked immunosorbent assay (plasmonic ELISA) is proposed initially for the application of CD81 in early screening for PE. In this work, a novel chromogenic substrate [(HAuCl4)-(N-methylpyrrolidone)-(Na3C6H5O7)] is designed based on the H2O2 dual catalysis reduction pathway of Au ions. The two reduction pathways of Au ions are controlled by H2O2 which ensures that the synthesis and growth of AuNPs are sensitive to H2O2. The amount of H2O2 correlates with the concentration of CD81 and directs the production of different-sized AuNPs in this sensor. Blue solutions are generated when analytes are present. When analytes are absent, solutions turn red. Therefore, due to different absorption peaks in red and blue, bimodal detection can be performed, and then two detection signals can be generated, one on signal at 550 nm and another off signal at 600 nm. This method exhibits a linear response to the logarithmic CD81 concentrations in the range of 0.1-1000 pg mL-1 with detection limits of 86 fg mL-1 and 152 fg mL-1 at two wavelengths. The false positive rate is low due to the nonspecific coloration caused by serum, which produces a more intense color contrast. The results indicate that the proposed dichromatic sensor could be used as a visual sensing platform for the direct detection of CD81 in biological samples and demonstrate its potential in preeclampsia diagnosis.

Shape dependent sensing potential of gold nanoparticles in etching based multicolorimetric plasmonic-ELISA.

In the present study, a systematic investigation has been carried out for the first time to assess the potential of three different shapes of gold nanoparticles (AuNPs), viz. nanorods (AuNRs), nanotriangles (AuNTs), and nanospheres (AuNSs), to develop a horseradish peroxidase (HRP) enzyme-mediated etching-based plasmonic ELISA (p-ELISA) strategy. The etching of the AuNPs in ELISA is achieved by 3′-3-5′-5-tetramethylbenzidine (TMB2+), which is produced by the biocatalytic conversion of chromogenic TMB via HRP. All three types of AuNPs were interacted with varying concentrations of TMB2+ (7–131 μM) (product of HRP enzyme reaction) and characterized for visible color change and by UV-Vis spectroscopy and transmission electron microscopy (TEM). From the comparative analysis of all three shapes of AuNPs, AuNRs exhibited vivid visible color change and absorbance intensity change compared to spherical and triangle-shaped nanoparticles. The TEM analysis of the etched nanoparticles revealed the gradual etching pattern of AuNRs compared to AuNTs which resulted in multicolor generation as opposed to AuNTs where the etching was relatively very fast and thus shows a faster shape transformation and poor color discrimination. Further, the potential of the AuNR etching-based optimized strategy was successfully demonstrated to develop an indirect competitive p-ELISA for human IgG detection. The developed p-ELISA showed an ultra-low visual limit of detection of 1 fg mL−1 (∼6.54 aM) without the aid of any sophisticated instruments. In the future, the developed competitive p-ELISA strategy can be easily employed to develop cost-effective, portable, and point-of-care assays for the detection of various disease biomarkers with ultra-high sensitivity.

Highly sensitive detection of salbutamol by ALP-mediated plasmonic ELISA based on controlled growth of AgNPs

Salbutamol (SAL), a short-acting β-2 adrenergic receptor agonist, is an undesirable addition to livestock farming and used to improve the growth rates. A direct competitive colorimetric immunoassay for SAL was developed. The alkaline phosphatase (ALP)-mediated controlled growth of the prereduction silver nanoparticles (AgNPs) was used to achieve the highly sensitive, colorimetric detection of SAL through the plasmonic ELISA. Click chemistry reaction was used to synthesize salbutamol biotin label (SAL-Bio) to achieve signal amplification and specific binding with antibody. ALP can efficiently hydrolyze sodium L-ascorbyl-2-phosphate and generate L-ascorbic acid, thereby reducing Ag+ and forming AgNPs. The plasmon resonance absorption signals of AgNPs were enhanced along with a distinct color change. The limit of detection of SAL can be decreased to 26.14 pg/mL. The method offers a good technique for the detection of many small molecular contaminants, such as pesticide residues and veterinary drug residues in food and edible water.

Silver nanoprism-based plasmonic ELISA for sensitive detection of fluoroquinolones.

Fluoroquinolones are synthetic antibiotics that are commonly used in animal husbandry, and the consumption of animal products with fluoroquinolone residues has imposed a serious threat to human health. Here, we report a plasmonic enzyme-linked immunosorbent assay (pELISA) method based on oxidative etching of silver nanoprisms (AgNPRs) for the quantitative and qualitative detection of danofloxacin (DAN), a fluoroquinolone antibiotic. AgNPRs that undergo colorimetric changes upon oxidative etching by H2O2 serve as the signal transducer in our design. An indirect competitive pELISA was constructed by introducing biotinylated monoclonal antibody (mAb), streptavidin and biotinylated glucose oxidase, which catalyzes the generation of H2O2 for etching AgNPRs. The quantitative detection limit of the proposed method was 0.24 ng mL-1 for DAN. The qualitative detection limit for DAN reached 0.32 ng mL-1, which was 32-fold lower than that of the assay using 3,3',5,5'-tetramethylbenzidine (TMB) as the signal transducer. The average recoveries of DAN in milk ranged from 103% to 121%, with a coefficient of variation of 0.6-3.41%. The recovery results were further confirmed using liquid chromatography-tandem mass spectrometry. In summary, the proposed AgNPR-etching pELISA exhibits high sensitivity, good accuracy and excellent reliability for the quantitative and qualitative detection of DAN in milk.

Plasmonic ELISA based on DNA-directed gold nanoparticle growth for Cronobacter detection in powdered infant formula samples

The traditional gold nanoparticle (AuNP) growth-based plasmonic ELISA (pELISA) strictly and directly controlled by reducing reagents can achieve high sensitivity, but it remains fragile toward the surrounding environment. This work developed a sandwich pELISA for Cronobacter detection in powdered infant formula samples by mediating AuNP growth through DNA. In this assay, DNA adsorbed on the surface of gold nanoseeds guided the anisotropic crystal growth with hydroxylamine as a reducing reagent, and the catalase-hydrogen peroxide (Cat-H2O2) system was introduced to bridge the DNA-directed AuNP growth and pELISA, as such DNA can be cleaved into fragments by the hydroxyl radical generated from oxidation of H2O2 through Fenton reagents. Under optimized conditions, the proposed pELISA can qualitatively detect Cronobacter species (Cronobacter muytjensii ATCC 51329) by the naked eye with a cut-off limit of 3 × 105 cfu/mL. This method also revealed a good linear range (3 × 102 to 3 × 107 cfu/mL) for quantitative detection of C. muytjensii ATCC 51329 with a limit of detection of 1.6 × 102 cfu/mL, which is approximately 162.5 times lower than that of horseradish peroxidase-based conventional ELISA (2.6 × 104 cfu/mL). By taking advantage of highly stable DNA-directed AuNP growth, the proposed method shows a good performance in powdered infant formula samples spiked with different concentrations of C. muytjensii ATCC 51329 with average recoveries ranging from 90.79 to 119.09% and coefficient of variation ranging from 4.24 to 9.55%. These values corresponded to an acceptable accuracy and precision for the proposed method. In brief, this work shows potential for screening other analytes in food safety, clinical diagnostics, and environmental monitoring.

A Gold Growth-Based Plasmonic ELISA for the Sensitive Detection of Fumonisin B1 in Maize.

In this paper, a highly sensitive plasmonic enzyme-linked immunosorbent assay (pELISA) was developed for the naked-eye detection of fumonisin B1 (FB1). Glucose oxidase (GOx) was used as an alternative to horseradish peroxidase as the carrier of the competing antigen. GOx catalyzed the oxidation of glucose to produce hydrogen peroxide, which acted as a reducing agent to reduce Au3+ to Au on the surface of gold seeds (5 nm), This reaction led to a color change in the solution from colorless to purple, which was observable to the naked eye. Various parameters that could influence the detection performance of pELISA were investigated. The developed method exhibited a considerably high sensitivity for FB1 qualitative naked-eye detection, with a visible cut-off limit of 1.25 ng/mL. Moreover, the proposed pELISA showed a good linear range of 0.31–10 ng/mL with a half maximal inhibitory concentration (IC50) of 1.86 ng/mL, which was approximately 13-fold lower than that of a horseradish peroxidase- (HRP)-based conventional ELISA. Meanwhile, the proposed method was highly specific and accurate. In summary, the new pELISA exhibited acceptable accuracy and precision for sensitive naked-eye detection of FB1 in maize samples and can be applied for the detection of other chemical contaminants.

Prereduction-promoted enhanced growth of silver nanoparticles for ultrasensitive colorimetric detection of alkaline phosphatase and carbohydrate antigen 125

A highly sensitive colorimetric method for detection of alkaline phosphatase (ALP) and carbohydrate antigen (CA125) was developed, which was achieved by ascorbic acid (AA) - mediated enhanced growth of silver nanoparticles (AgNPs) promoted with NaBH4 as pre-reducing agent. With prereduction by NaBH4, the AA controlled growth of AgNPs was promoted and the localized surface plasmon resonance absorbance was much higher compared with that without prereduction. ALP could hydrolyze L-ascorbic acid 2-phosphate trisodium salt (AAP) to form AA, which could then reduce Ag+ to AgNPs resulting in the enhancement of localized surface plasmon resonance peak of AgNPs and accompanying the color change from colorless to bright yellow. Benefiting from the highly sensitive response of Ag+ to AA, the detection limit for ALP could be lowered to 0.003 U L−1, and the proposed plasmonic ELISA could achieve a low detection limit of 1.75 U mL−1 for CA125. Moreover, this method was validated for the analysis of ALP and CA125 in human serum samples, giving results matched well with conventional method, demonstrating the potential application of the developed method in clinical diagnosis.

Plasmonic ELISA based on enzyme-assisted etching of Au nanorods for the highly sensitive detection of aflatoxin B1 in corn samples

In this work, we reported an advanced enzyme-assisted etching method using gold nanorods (AuNRs) as signal output of plasmonic enzyme-linked immunosorbent assay (pELISA) for the quantitative or qualitative detection of aflatoxin B1 (AFB1) in real corn samples. AFB1-labeled glucose oxidase was adopted as the competing antigen to catalyze glucose into H2O2. Meanwhile, horseradish peroxidase was employed to catalyze H2O2 and hence generate .OH. The AuNRs with the aspect ratio of 2:1 were chemically etched by OH to a rod-like morphology. This occurrence reduced the optical density at 650 nm and resulted in a vivid color response from bluish-green to red easily detectable by a microplate reader for quantitative AFB1 analysis or by the naked eye for qualitative AFB1 detection. Various parameters that may influence the detection performance of pELISA were investigated. The proposed method showed an extremely high sensitivity for qualitative AFB1 detection, with a visible cut-off value of 12.5 pg/mL. The technique also achieved a good linear range of 3.1–150 pg/mL for quantitative AFB1 analysis, with a half-maximal inhibitory concentration of 22.3 pg/mL, which was approximately 32 folds lower than those of conventional ELISA (707 pg/mL). The average recoveries for corn samples spiked with different concentrations of AFB1 ranged from 82% to 115%, with a coefficient of variation that ranged from 2% to 13%. These values corresponded to an acceptable accuracy and precision for the proposed method. In addition, the reliability of the proposed method was further confirmed by the liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. In brief, this work offers an improved screening strategy with high sensitivity and robustness for the qualitative or quantitative detection of mycotoxins or other pollutants in food safety monitoring.

Plasmonic ELISA for naked-eye detection of ochratoxin A based on the tyramine-H2O2 amplification system

Abstract A novel direct competitive plasmonic enzyme-linked immunosorbent assay (dc-pELISA) was applied to detect ochratoxin A (OTA) with naked eyes. In this assay, horseradish peroxidase (HRP) + hydrogen peroxide (H2O2) + tyramine (TYR)-induced gold nanoparticle (AuNP) aggregation was considered as a signal output; AuNP aggregation could be triggered through the phenol polymerization of TYR, which was induced by hydroxyl radicals from HRP-catalyzed H2O2; OTA-labeled catalase (CAT) was used as a competing antigen to consume H2O2. Owing to the combined advantages of ultrahigh CAT catalytic activity for H2O2 and dual-color responses (red and blue) generated through AuNP aggregation, the proposed method was highly sensitive and thus could be employed with naked eyes to detect OTA qualitatively with a cut-off limit of 150 pg/mL. Our method also demonstrated a good dynamic linear range (12.5–150 pg/mL) for quantitative OTA determination with a reliable correlation coefficient of R2 = 0.992, a half-maximal inhibitory concentration of 84.75 pg/mL, and a detection limit of 17.8 pg/mL. In brief, this newly-designed technique is considerably suitable for high-throughput screening detection or point-of-care diagnostics in resource-constrained regions because of the easy readout of results by naked eyes without the use of advanced detection equipment.

A plasmonic ELISA for the naked-eye detection of chromium ions in water samples.

Here, we describe the development of a triangular silver nanoprism (AgNPR) etching-based plasmonic ELISA for the colorimetric determination of Cr(III) levels in environmental water samples. This involved the creation of a novel signal generation system (substrate reaction solution) for a competitive ELISA in which hydrogen peroxide (H2O2) is used to etch triangular AgNPRs, inducing a change in color. This is achieved by controlling the H2O2 concentration that remains after degradation by catalase, which is conjugated to the secondary antibody of the ELISA. Because the degree of color change and the shift in the absorption spectrum of the substrate reaction solution are closely correlated with the Cr(III) concentration, this plasmonic ELISA can be used not only for the quantification of Cr(III) concentrations ranging from 3.13 to 50ng/mL, with a limit of detection (LOD) of 3.13ng/mL, but also for the visual detection (indicated by a color change from blue to mauve) of Cr(III) with a sensitivity of 6.25ng/mL by the naked eye. Therefore, the plasmonic ELISA developed in this work represents a new strategy for heavy metal ion detection and has high potential applicability in resource-constrained areas. Graphical Abstract Schematic diagram of triangular silver nanoprism etching-based signal generation system.

Dual-color plasmonic enzyme-linked immunosorbent assay based on enzyme-mediated etching of Au nanoparticles.

Colorimetric enzyme-linked immunosorbent assay utilizing 3′-3-5′-5-tetramethylbenzidine(TMB) as the chromogenic substrate has been widely used in the hospital for the detection of all kinds of disease biomarkers. Herein, we demonstrate a strategy to change this single-color display into dual-color responses to improve the accuracy of visual inspection. Our investigation firstly reveals that oxidation state of 3′-3-5′-5-tetramethylbenzidine (TMB2+) can quantitatively etch gold nanoparticles. Therefore, the incorporation of gold nanoparticles into a commercial TMB-based ELISA kit could generate dual-color responses: the solution color varied gradually from wine red (absorption peak located at ~530 nm) to colorless, and then from colorless to yellow (absorption peak located at ~450 nm) with the increase amount of targets. These dual-color responses effectively improved the sensitivity as well as the accuracy of visual inspection. For example, the proposed dual-color plasmonic ELISA is demonstrated for the detection of prostate-specific antigen (PSA) in human serum with a visual limit of detection (LOD) as low as 0.0093 ng/mL.

Nanospherical Brush as Catalase Container for Enhancing the Detection Sensitivity of Competitive Plasmonic ELISA.

Plasmonic enzyme-linked immunosorbent assay (pELISA) based on catalase (CAT)-mediated gold nanoparticle growth shows great potential for the determination of disease-related biomarkers at ultralow concentrations by using sandwich formats. However, the relatively low sensitivity of this strategy using competitive formats limits its adoption for hapten detection. Herein, we present an improved competitive pELISA for ultrasensitive detection of ochratoxin A (OTA), where silica nanoparticles carrying poly(acrylic acid) brushes (SiO2@PAA) were used to decrease the affinity of competing antigens to anti-OTA monoclonal antibodies and amplify the signal as a "CAT container" (SiO2@PAA@CAT). The developed competitive pELISA exhibits extremely high sensitivity for OTA with detection limits of 10(-18) and 5 × 10(-20) g/mL by the naked eye and microplate reader, respectively. These values are at least 7 orders of magnitude lower than that of competitive CAT-based pELISA (10(-11) g/mL by the naked eye) and 8 orders of magnitude lower than that of horseradish peroxidase-based conventional ELISA (10(-11) g/mL by the microplate reader), respectively. Reliability and robustness of the proposed method were evaluated using actual agricultural products and human serum samples. This study demonstrated the potential of this modified method in practical applications involving the ultrasensitive detection of mycotoxins or other haptens.

Plasmonic ELISA based on the controlled growth of silver nanoparticles.

Herein, we demonstrate a plasmonic ELISA based on the alkaline phosphatase (ALP)-mediated growth of silver nanoparticles (AgNPs) for the sensitive, rapid, and naked-eye detection of cancer biomarkers in clinical serum samples. This approach was used to measure the low-abundance alpha fetal protein (AFP) in clinical sera, which demonstrates its great capability in the differentiation of cancers and evaluation of therapeutic responses. Impressively, the readout of the plasmonic assay depends on the rapid formation of Ag colloidal solutions with various degrees of yellow color, which can be distinguished by the naked eye, without the need for sophisticated platforms. The limit of detection of the plasmonic ELISA for alpha fetal protein (AFP) can be as low as 0.23 ng mL-1, which is approximately 10 folds lower than that of conventional ELISA. This plasmonic ELISA opens a new avenue for the early detection of cancers and monitoring of cancer reoccurrence especially in resource-poor regions where convenient diagnostic tools are highly desirable.

Silver nanoprism etching-based plasmonic ELISA for the high sensitive detection of prostate-specific antigen.

Ultrasensitive and quantitative detection using simple and low-cost assays is critical in clinical diagnostics. In this report, we developed a triangular silver nanoprism (AgNPRs) etching-based plasmonic biosensor for the detection of cancer biomarkers. The triangular AgNPRs-based plasmonic biosensor is an enzyme-linked immunosorbent assay combined with the enzyme-mediated surface plasmon resonance (SPR) of triangular AgNPRs. Triangular AgNPRs uses the immune response of prostate-specific antigen (PSA) to trigger the glucose oxidase (GOx)-catalysed oxidation of glucose (Glu), producing hydrogen peroxide. Hydrogen peroxide acts as an oxidant to etch the triangular AgNPRs into smaller spherical silver nanoparticles, which is accompanied by a substantial blueshift of the SPR peak and a colourimetric blue-to-purple change that can be observed by the naked eye. The SPR peak shift enables the quantitative assessment of PSA due to the remarkable colour change. The triangular AgNPRs-based plasmonic ELISA approach exhibited a quasilinear response to logarithmic PSA concentrations in the range of 10fg/mL to 100pg/mL with a limit of detection (LOD) of 4.1fg/mL. In addition, the LOD of PSA in this approach exceeds that of the conventional HRP-based ELISA (1.25ng/mL) approach by more than 5 orders of magnitude. Patient serum samples from 16 donors were assayed with triangular AgNPRs-based plasmonic ELISA. The results from the triangular AgNPRs-based immunoassay and the time-resolved fluorescence immunoassay showed excellent correlation, and there were no significant differences in the quantified amounts of PSA. The triangular AgNPRs-based plasmonic ELISA approach has advantages (ultrasensitive, cost-effective, ease of operation) that are expected to be of great interest in diagnostics and to be suitable for a point-of-care test.

Highly sensitive “signal on” plasmonic ELISA for small molecules by the naked eye

Based on the H2O2-mediated generation of gold nanoparticles, a colorimetric catalase nanosensor was developed and utilized as a signal output in a catalase-labelled immunoassay. Methyltestosterone (MT), which is used illegally as a growth promoter during the cattle production cycle, was selected as a small molecule model in this new ELISA development. As a result, a “signal on” plasmonic ELISA for MT was established. This plasmonic ELISA can quantitatively detect MT by spectrophotometry with a 50 times higher sensitivity than the traditional ELISA. In addition, it allows the highly sensitive and easily qualitative detection of MT by the naked eye. This plasmonic direct competitive ELISA can be utilized for developing cost-effective and sensitive ELISA against various small molecules by the naked eye.

Plasmonic ELISA for the ultrasensitive detection of Treponema pallidum

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 1pg/mL–10ng/mL with a detection limit of 0.98pg/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.