Immunosensors using metallic nanoparticle-based signal enhancement for bacterial detection and tuberculosis diagnosis

Escherichia coli O157:H7 is one of the main foodborne/waterborne bacterial pathogens that can cause human illnesses with threat to public health. To control the spread of the contaminated food/water and minimize the harm to public health, rapid and sensitive detection methods need to be implemented. However, standard culture method requires two to four days to obtain results. The application of nanomaterials has drawn interest in the biosensor research to develop timely and low cost detection systems. Because of their unique characteristics, nanoparticles have been used to enhance biosensor sensitivity by increasing the target molecule capture efficiency or by amplifying detection signals. In this dissertation research, nanoparticle-based biosensors were designed for the rapid detection of E. coli O157:H7 in broth. Magnetic nanoparticles (MNPs) were conjugated with monoclonal antibodies to separate target E. coli O157:H7 cells from samples. Gold nanoparticles (AuNPs) conjugated with polyclonal antibodies were then introduced to the MNP-target complexes to form MNP-target-AuNP. By measuring the amount of gold nanoparticles through an electrochemical method, the presence and the amount of the target bacteria were determined. Based on this biosensor using AuNPs as labels for signal amplification, a tri-nano electrochemical immunosensor was developed by using three nanoparticles for the rapid detection. The gold nanoparticles (AuNPs) were conjugated with lead sulfide (PbS) nanoparticles as electrochemical reporters via oligonucleotide linkage. AuNPs were also functionalized with polyclonal anti-E. coli O157:H7 antibodies in order to bind the target bacterial cells which were captured and separated from the sample by antibody-functionalized MNPs. Because each AuNP was linked to multiple PbS nanoparticles, each binding event to the target resulted in substantial amplification. The signal of PbS was measured on screen-printed carbon electrode (SPCE) by square wave anodic stripping voltammetry (SWASV). Results showed that the biosensor could detect E. coli O157:H7 in the range of 101 to 106 colony forming units per milliliter (cfu/ml) with a signal-to-noise ratio ranging from 2.77 to 4.31. With sample preparation being minimized, results were obtained within 1 h from sample processing to final readout. Tuberculosis (TB) is considered as one of the most widely spread infectious diseases, with estimated 8.8 million new cases and 2 million deaths annually. The biosensor developed in this dissertation research was also applied for TB diagnosis. Gold nanoparticles with anti-IFN-gamma antibody were conjugated to oligonucleotides terminated with cadmium sulfide (CdS) nanoparticles. At the same time, AuNPs were conjugated with anti-IP-10 antibody and oligonucleotides terminated with PbS nanoparticles. Therefore, the electrochemical signals of cadmium and lead indicated IFN-gamma and IP-10, respectively. By introducing MNPs with antibodies to IFN-gamma or IP-10 and AuNP conjugates, IFN-gamma and IP-10 were detected separately in buffer and simultaneously in both buffer and plasma. The results showed that IFN-gamma in the range of 0.01 IU/ml to 10 IU/ml and IP-10 in the range of 0.01 ng/ml to 100 ng/ml were detected in 1 h. Due to its rapidity, high sensitivity and multiplex detection capability, this tri-nanobiosensor has potential applications in public health, biodefense, and food/water safety monitoring.