Rapid, Low‑Cost LSPR Nanobiosensor for Accurate Brucellosis Diagnosis

Abstract

Brucellosis remains the most common bacterial zoonosis worldwide. Conventional diagnostics—culture, serology, and PCR—suffer from long turnaround times, limited sensitivity, or cross‑reactivity. Here we present a gold‑nanoparticle localized surface plasmon resonance (LSPR) nanobiosensor that overcomes these shortcomings. Smooth lipopolysaccharides (LPS) from Brucella melitensis and Brucella abortus were covalently immobilized on 10‑nm gold nanoparticles. Binding of patient anti‑Brucella antibodies produced a measurable red‑shift in the LSPR peak, enabling quantitative discrimination between infected and uninfected sera. In a blinded study of 40 clinical samples (20 culture‑positive, 20 culture‑negative), the sensor achieved 85 % sensitivity, 100 % specificity, 100 % positive predictive value, and 86 % negative predictive value—comparable to or exceeding standard serologic assays. The assay requires only 30 min, uses inexpensive reagents, and is amenable to point‑of‑care deployment.

Introduction

Brucella species are slow‑growing, Gram‑negative coccobacilli that infect a broad range of domestic and wild animals. Among the 12 recognized species, B. melitensis, B. abortus, B. suis, and B. canis are the principal human pathogens, with B. melitensis being the most virulent. Human brucellosis presents with nonspecific flu‑like symptoms, making laboratory confirmation essential. Traditional culture, although the gold standard, requires 5–7 days and carries biosafety risks. Serologic assays suffer from cross‑reactivity and cannot distinguish active from past infection. Nucleic‑acid amplification offers speed but may remain positive long after cure. Thus, a rapid, accurate, and affordable diagnostic tool is urgently needed.

Localized surface plasmon resonance (LSPR) exploits the collective oscillation of conduction electrons in metallic nanoparticles, generating a sharp absorbance peak that shifts upon biomolecular binding. Gold nanoparticles (AuNPs) of ~10 nm size offer high surface‑to‑volume ratio and stable optical properties, making them ideal LSPR transducers. By coupling LPS antigens to AuNPs, the LSPR sensor can detect anti‑Brucella antibodies with high sensitivity and specificity in a simple colorimetric readout.

Methods

Bacterial culture and LPS extraction

Smooth strains of Brucella melitensis and Brucella abortus were grown on Luria–Bertani agar. LPS was extracted via a hot‑phenol method, purified by silver‑stained SDS‑PAGE, and quantified with 1,9‑dimethyl methylene blue against a Salmonella typhimurium standard. Protein contamination was ruled out by the Bradford assay and nucleic acid content was <0.2 % of LPS mass.

Gold nanoparticle synthesis

AuNPs were produced by the Turkevich reduction of HAuCl₄ with sodium citrate, followed by sodium borohydride addition at room temperature. The resulting colloid exhibited a 530 nm plasmon peak and an average diameter of 10 nm, confirmed by SEM and dynamic light scattering (DLS). Zeta potential of −28 mV indicated good colloidal stability.

Nanoprobe preparation

AuNPs were functionalized with thioglycolic acid (TGA) for carboxyl surface groups. Optimal TGA incubation was 24 h, after which 300 µg/ml LPS was covalently coupled via EDC/NHS chemistry. The final probe retained a strong 530 nm absorbance peak.

Antibody detection

Serum samples were diluted 1:50 in PBS and mixed 1:2 (100 µl serum + 200 µl probe). After 30 min incubation, the mixture was centrifuged and the supernatant’s absorbance measured. Red‑shifts of ≥4.38 nm, defined as the cutoff (mean + 2 SD of negative controls), were considered positive.

Ethics

All sera were collected with informed consent and IRB approval (IR.FUMS.1396.324). Participant identities were coded.

Validation

Forty sera (20 culture‑positive, 20 culture‑negative) were tested. Results were compared to culture, standard tube agglutination test (SAT), and commercial ELISA kits for IgM/IgG. Sensitivity, specificity, PPV, and NPV were calculated per standard formulas.

Statistical analysis

Data normality was assessed by Kolmogorov–Smirnov; ANOVA compared groups. Analyses were performed with GraphPad Prism 8 and SPSS 9.

Results

LPS extraction

SDS‑PAGE confirmed a ~1 % yield (wet weight) with negligible protein and nucleic acid contamination.

AuNP characterization

SEM images showed uniform 10 nm spheres. DLS indicated narrow size distribution (10 ± 2 nm). Zeta potential was −28 mV. UV–Vis peak at 530 nm confirmed successful synthesis.

Probe optimization

24 h TGA coating produced maximal functionalization without spectral shift. LPS loading plateaued at 300 µg/ml, beyond which no further absorbance change occurred.

Proof of concept

Incubation with positive anti‑Brucella control antibodies produced a clear red‑shift, whereas negative controls did not.

Cutoff determination

Mean red‑shift in negatives was 1.70 nm; positives averaged 6.08 nm. A 4.38 nm cutoff yielded 5 % false positives and 0 % false negatives in the validation set.

Diagnostic performance

Compared to culture, the LSPR assay showed 85 % sensitivity, 100 % specificity, 100 % PPV, and 86 % NPV. SAT achieved 90 % sensitivity, 100 % specificity; ELISA IgM/IgG had lower PPV (90 % and 81 % respectively).

Discussion

Brucellosis causes ~500,000 new human cases annually. The LSPR nanobiosensor offers a rapid (≤30 min), inexpensive (<$1 per test), and highly specific alternative to conventional methods. By detecting anti‑LPS antibodies via a measurable spectral shift, the assay circumvents the time‑consuming culture and the cross‑reactivity issues of serology. Although it provides qualitative rather than quantitative antibody levels, the high PPV and NPV make it suitable for both screening and confirmation in endemic regions.

Limitations include the persistence of antibodies post‑cure, which may yield positive results in recovered patients, and the need for a spectrophotometer for precise shift measurement. Future work will explore portable, handheld readers to enable true point‑of‑care deployment.

Conclusion

The 10‑nm gold‑nanoparticle LSPR biosensor demonstrates rapid, accurate, and cost‑effective detection of anti‑Brucella antibodies. Its high specificity and comparable sensitivity to standard serology, coupled with minimal equipment requirements, make it a compelling tool for diagnosing brucellosis, especially in resource‑limited settings.

Availability of data and materials

Datasets are available from the corresponding author upon reasonable request.

Abbreviations

LPS

Lipopolysaccharides

GNPs

Gold nanoparticles

DLS

Dynamic light scattering

LSPR

Localized surface plasmon resonance

SAT

Standard tube agglutination test

ELISA

Enzyme‑linked immunosorbent assay

PPV

Positive predictive value

NPV

Negative predictive value

PSPR

Propagating surface plasmon resonance

SDS‑PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SEM

Scanning electron microscopy

TGA

Thioglycolic acid

NHS

N‑hydroxysuccinimide

EDC

N‑(3‑dimethylaminopropyl)‑N′‑ethylcarbodiimide hydrochloride

PBS

Phosphate‑buffer saline