Allen D. Taylor

Functionalizable surface platform with reduced nonspecific protein adsorption from full blood plasma—Material selection and protein immobilization optimization

In this work, zwitterionic polymers are investigated as ultra-low fouling and functionalizable coatings for biosensors, nanoparticle-based diagnostics, and microarrays to enable detections in real-world complex media. The effect of the spacer length between the two charged groups on the nonfouling properties of zwitterionic poly(carboxybetaine acrylamide) (polyCBAA) was studied in blood plasma and serum. The polyCBAA polymer with an ethylene spacer was selected for protein immobilization studies. A polyCBAA-coated surface was functionalized with antibodies using a simple and fast amino coupling chemistry for direct protein immobilization in two simple steps: surface activation and protein immobilization/background deactivation. The effect of pH was found to be very important for both steps and it was optimized. The functionalized polyCBAA surface exhibited very low fouling properties even when exposed to undiluted blood plasma for more than 6h with <7ng/cm(2) of adsorbed proteins. The biological activity of the immobilized proteins was demonstrated with the detection of a model protein in undiluted blood plasma. A recently developed highly sensitive four-channel surface plasmon resonance (SPR) sensor was used for the evaluation of specific and nonspecific protein adsorption to these surfaces.

Comparative study of SPR and ELISA methods based on analysis of CD166/ALCAM levels in cancer and control human sera

Surface plasmon resonance (SPR), as a label free method for analysis of various analytes, has significantly advanced in recent years. However, assessment of the performance of SPR compared to label-based immunoassays such as the commonly used multiplexed enzyme-linked immunosorbent assay (ELISA) is limited, particularly for applications involving complex media. In this work, an optimized SPR assay was implemented and its performance compared with an ELISA assay for CD166/activated cell leukocyte adhesion molecule (ALCAM), as candidate pancreatic cancer marker, based on direct and amplified detection in buffer and in human serum samples from healthy individuals and subjects with cancer. ALCAM antibody was immobilized on the surface of a four-channel SPR sensor via physical adsorption onto charged amine-terminated alkanethiolates to mimic the ELISA plate surface. Excellent correlations between SPR and ELISA results were achieved in buffer and in human serum. SPR detected the target protein with a similar sensitivity to sandwich ELISA, with a detection limit below ng/mL. The detection time, sample consumption, throughput, signal referencing, and surface blocking and washing for detection in human serum were evaluated. It is demonstrated that SPR can distinguish between ALCAM levels in cancer and control sera using direct detection without the need for additional amplification steps.

Direct detection of carcinoembryonic antigen autoantibodies in clinical human serum samples using a surface plasmon resonance sensor

The ability to detect biomarkers in human serum is important for cancer diagnostics. The work presented focuses on the establishment of a surface plasmon resonance (SPR) biosensor as a means for detecting varying levels of autoantibody biomarkers in human serum samples. Carcinoembryonic antigen (CEA) is a biomarker that is present in human serum. It is thought that CEA levels become elevated in patients with colon and ovarian cancer, causing a corresponding increase in the autoantibody level in human serum. Detection of this CEA autoantibody increase could be used to diagnose cancer in patients. Using a SPR biosensor, human serum samples were screened directly for CEA antibody levels. Results using a sandwich assay with a SPR sensor demonstrated the same linear trend as seen from an established enzyme-linked immunosorbent assay (ELISA) method. Serum samples from five healthy individuals were used to establish a threshold value for differentiating a cancerous serum sample from a negative sample with a 95% confidence. Three serum samples from cancer patients with positive CEA antibody levels as evaluated by ELISA were used to test the criterion.

Hybrid surface platform for the simultaneous detection of proteins and DNAs using a surface plasmon resonance imaging sensor.

In this work, we present a novel surface and assay for the simultaneous detection of DNA and protein analytes on a surface plasmon resonance (SPR) imaging sensor. A mixed DNA/oligo (ethylene glycol) (OEG) self-assembled monolayer (SAM) is created using a microarrayer. Thiol-modified single-stranded DNA sequences are spotted onto a gold-coated glass substrate. Backfilling with an OEG-modified alkanethiol creates a protein-resistant surface background. Antibodies conjugated to complementary single-stranded DNA sequences are immobilized on the surface through DNA hybridization. By converting only part of the DNA array into a protein array, simultaneous detections of DNA and protein analytes are possible. A model system of two cDNA sequences and two human pregnancy hormones are used to demonstrate the assay. No cross-reactivity was observed between DNA or protein analytes and nontargeted immobilized cDNA sequence or antibodies. A response from a detection of a single analyte in a mixture of protein and DNA analytes corresponds well with that of a single-analyte solution.

Directly functionalizable surface platform for protein arrays in undiluted human blood plasma.

Protein arrays are a high-throughput approach for proteomic profiling, vital for achieving a greater understanding of biological systems, in addition to disease diagnostics and monitoring therapeutic treatments. In this work, zwitterionic carboxybetaine polymer (pCB) coated substrates were investigated as an array surface platform to enable convenient amino-coupling chemistry on a single directly functionalizable and unblocked film for the sensitive detection of target analytes from undiluted human blood plasma. Using a surface plasmon resonance (SPR) imaging sensor, the antibody immobilization conditions which provided excellent spot morphology and the largest antigen response were determined. It was found that pCB functionalization and the corresponding antigen detection both increased with pH and antibody concentration. Additionally, immobilization only required an aqueous buffer without the need for additives to improve spot quality. The nonspecific protein adsorption to undiluted human plasma on both the antibody immobilized pCB spots and the background were found to be about 9 and 6 ng/cm(2), respectively. A subsequent array consisting of three antibodies spotted onto pCB revealed little cross-reactivity for antigens spiked into the undiluted plasma. The low postfunctionalized nonfouling properties combined with antibody amplification showed similar sensitivities achievable with conventional spectroscopic SPR sensors and the same pCB films, but now with high-throughput capabilities. This represents the first demonstration of low fouling properties following antibody functionalization on protein arrays from undiluted human plasma and indicates the great potential of the pCB platform for high-throughput protein analysis.

Surface Plasmon Resonance (SPR) Sensors for the Detection of Bacterial Pathogens

Modern biosensor technologies can provide rapid quantification of bacterial pathogens. Surface plasmon resonance (SPR) sensors are an optical platform capable of highly sensitive and specific measuring of biomolecular interactions in real-time. This label-free technology can quantify the kinetics, affinity and concentration of surface interactions. SPR sensors have been used to detect bacterial pathogens in clinical and food-related samples. This chapter discusses the fundamental theory behind SPR sensors and state-of-the-art SPR instrumentation, surface chemistries, molecular recognition elements and detection strategies, as well as specific challenges associated with bacterial detection using SPR sensors. SPR-based detections of bacterial cells, genetic markers and antibody biomarkers are reviewed and discussed.

Tetrodotoxin Detection by a Surface Plasmon Resonance Sensor in Pufferfish Matrices and Urine

Tetrodotoxin (TTX) poisoning is most commonly associated with consumption of pufferfish. TTX is a low molecular weight (~319 Da) neurotoxin that selectively blocks voltage-sensitive Na

SPR Biosensors for Food Safety

2 Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 2.1 Escherichia Coli O157:H7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 2.2 Salmonella spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 2.3 Listeria Monocytogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 2.4 Campylobacter Jejuni . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 2.5 Clostridium Perfringens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 2.6 Yersinia Enterocolitica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 2.7 Multiple Bacteria Detections . . . . . . . . . . . . . . . . . . . . . . . . . . 214