Novel Vertical Flow Immunoassay with Au@PtNPs for Rapid, Ultrasensitive, and On-Site Diagnosis of Human Brucellosis

Brucellosis is an infectious zoonosis caused by Brucella with clinical symptoms of wavy fever, fatigue, and even invasion of tissues and organs in the whole body, posing a serious threat to public health around the world. Herein, a novel vertical flow immunoassay based on Au@Pt nanoparticles (Au@PtNPs-VFIA) was established for detection of Brucella IgG antibody in clinical serum samples. The testing card of Au@PtNPs-VFIA was manufactured by printing the purified Brucella LPS and goat antimouse IgG on the nitrocellulose membrane as the test-spot or control-spot, respectively. Au@PtNPs labeled with protein G (Au@PtNPs-prG) were concurrently employed as detection probes presenting visible spots and catalysts mimicking catalytic enzymes to catalyze the DAB substrate (H2O2 plus O-phenylenediamine) for deepening color development. The testing procedure of Au@PtNPs-VFIA takes 2–3 min, and the limit of detection (LOD) for Brucella antibody is 0.1 IU/mL, which is faster and more sensitive than that of Au@PtNP-based lateral flow immunoassay (Au@PtNPs-LFIA: 15 min and 1.56 IU/mL, respectively). By comparing with vertical flow immunoassay based on classic Au nanoparticles (AuNPs-VFIA), the Au@PtNPs-VFIA is 32 times or 16 times more sensitive with or without further development of DAB substrate catalysis. Au@PtNPs-VFIA did not react with the serum samples of Gram-negative bacterium infections but only weakly cross-reacted with diagnostic serum of Y. enterocolitica O9 infection. In detection of clinical samples, Au@PtNPs-VFIA was validated for possessing 98.33% sensitivity, 100% specificity, and 99.17% accuracy, which were comparable with or even better than those obtained by the Rose-Bengal plate agglutination test, serological agglutination test, AuNPs-VFIA, and Au@PtNPs-LFIA. Therefore, this newly developed Au@PtNPs-VFIA has potential for rapid, ultrasensitive, and on-site diagnosis of human Brucellosis in clinics.