Yildiz Uludag

Cancer Biomarker Detection in Serum Samples Using Surface Plasmon Resonance and Quartz Crystal Microbalance Sensors with Nanoparticle Signal Amplification

Early detection of cancer is vital for the successful treatment of the disease. Hence, a rapid and sensitive diagnosis is essential before the cancer is spread out to the other body organs. Here we describe the development of a point-of-care immunosensor for the detection of the cancer biomarker (total prostate-specific antigen, tPSA) using surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) sensor platforms in human serum samples. K(D) of the antibody used toward PSA was calculated as 9.46 × 10(-10) M, indicating high affinity of the antibody used in developing the assay. By performing a sandwich assay using antibody-modified nanoparticles concentrations of 2.3 ng mL(-1) (Au, 20 nm) and 0.29 ng mL(-1) (8.5 pM) (Au, 40 nm) tPSA in 75% human serum were detected using the developed assay on an SPR sensor chip. The SPR sensor results were found to be comparable to that achieved using a QCM sensor platform, indicating that both systems can be applied for disease biomarkers screening. The clinical applicability of the developed immunoassay can therefore be successfully applied to patients serum samples. This demonstrates the high potential of the developed sensor devices as platforms for clinical prostate cancer diagnosis and prognosis.

Development of a sensitive detection method of cancer biomarkers in human serum (75%) using a quartz crystal microbalance sensor and nanoparticles amplification system.

A simple and sensitive sensor method for cancer biomarkers [prostate specific antigen (PSA) and PSA-alpha 1-antichymotrypsin (ACT) complex] analysis was developed, to be applied directly with human serum (75%) by using antibody modified quartz crystal microbalance sensor and nanoparticles amplification system. A QCM sensor chip consisting of two sensing array enabling the measurement of an active and control binding events simultaneously on the sensor surface was used in this work. The performance of the assay and the sensor was first optimised and characterised in pure buffer conditions before applying to serum samples. Extensive interference to the QCM signal was observed upon the analysis of serum. Different buffer systems were then formulated and tested for the reduction of the non-specific binding of sera proteins on the sensor surface. A PBS buffer containing 200 microg mL(-1) BSA, 0.5 M NaCl, 500 microg mL(-1) dextran and 0.5% Tween 20, was then selected which eliminated the interfering signal by 98% and enabled the biomarker detection assay to be performed in 75% human serum. By using Au nanoparticles to enhance the QCM sensor signal, a limit of detection of 0.29 ng mL(-1) PSA and PSA-ACT complex (in 75% serum) with a linear dynamic detection range up to 150 ng mL(-1) was obtained. With the achieved detection limit in serum samples, the developed QCM assay shows a promising technology for cancer biomarker analysis in patient samples.

Real-time and sensitive detection of Salmonella Typhimurium using an automated quartz crystal microbalance (QCM) instrument with nanoparticles amplification.

The accidental contamination of Salmonella in raw and processed foods is a major problem for the food industry worldwide. At present many of the currently used methods for Salmonella detection are time and labour intensive. Therefore, rapid detection is a key to the prevention and identification of problems related to health and safety. This paper describes the application of a new quartz crystal microbalance (QCM) instrument with a microfluidic system for the rapid and real time detection of Salmonella Typhimurim. The QCMA-1 bare gold sensor chip which contain two sensing array was modified by covalently immobilising the monoclonal capture antibody on the active spot and a mouse IgG antibody on the control spot using a conventional amine coupling chemistry (EDC-NHS). The binding of the Salmonella cells onto the immobilised anti-Salmonella antibody alters the sensor frequency which was correlated to cells concentration in the buffer samples. Salmonella cells were detected using direct, sandwich, and sandwich assay with antibody conjugated gold-nanoparticles. The performance of the QCM immunosensor developed with gold-nanoparticles gave the highest sensitivity with a limit of detection (LOD) ~10-20 colony forming unit (CFU) ml(-1) compared to direct and sandwich assay (1.83 × 10(2) CFU ml(-1) and 1.01 × 10(2) CFU ml(-1), respectively). The sensor showed good sensitivity and selectivity for Salmonella in the presence of other bacteria in real food samples and helped in reducing the pre-enrichment step, hence, demonstrating the potential of this technology for the rapid and sensitive microbial analysis.

Surface plasmon resonance based immunosensor for the detection of the cancer biomarker carcinoembryonic antigen

An immunoassay in optimised conditions with a highly sensitive surface plasmon resonance (SPR) based biosensor was developed for the detection of the cancer biomarker carcinoembryonic antigen (CEA). Different formats of the immunoassay were initially investigated on the surface of the gold sensor chip. A self-assembled monolayer (SAM) was formed on the gold chip using 11-mercaptoundecanoic acid (MUDA), before the immobilisation of the antibodies was conducted. The assay was then formed in a direct capture and a sandwich assay. In order to increase the sensor signal the CEA antigen was incubated with the detection/capture antibody before it was injected to the sensor chip surface and the results were recorded in real-time using the Biacore 3000 instrument. A detection limit of 3 ng ml(-1) CEA was obtained with a dynamic detection range from 3 ng ml(-1) to 400 ng ml(-1) with correlation coefficients of 1.00 and 0.99 for the sandwich and rabbit anti-mouse (RAM) capture assay. Kinetic data analysis was performed for the standard capture test and subsequently for the developed assays and R(max) showed an increase from 215 RU for the standard capture test to 428 RU for the RAM-capture assay and 734 RU for the sandwich assay, respectively. The developed SPR immunosensor using the sandwich assay format showed high sensitivity and reproducibility for CEA detection which makes it a promising procedure for cancer biomarker analysis.

Development of surface chemistry for surface plasmon resonance based sensors for the detection of proteins and DNA molecules

The immobilisation of biological recognition elements onto a sensor chip surface is a crucial step for the construction of biosensors. While some of the optical biosensors utilise silicon dioxide as the sensor surface, most of the biosensor surfaces are coated with metals for transduction of the signal. Biological recognition elements such as proteins can be adsorbed spontaneously on metal or silicon dioxide substrates but this may denature the molecule and can result in either activity reduction or loss. Self assembled monolayers (SAMs) provide an effective method to protect the biological recognition elements from the sensor surface, thereby providing ligand immobilisation that enables the repeated binding and regeneration cycles to be performed without losing the immobilised ligand, as well as additionally helping to minimise non-specific adsorption. Therefore, in this study different surface chemistries were constructed on SPR sensor chips to investigate protein and DNA immobilisation on Au surfaces. A cysteamine surface and 1%, 10% and 100% mercaptoundecanoic acid (MUDA) coatings with or without dendrimer modification were utilised to construct the various sensor surfaces used in this investigation. A higher response was obtained for NeutrAvidin immobilisation on dendrimer modified surfaces compared to MUDA and cysteamine layers, however, protein or DNA capture responses on the immobilised NeutrAvidin did not show a similar higher response when dendrimer modified surfaces were used.

Piezoelectric sensors based on molecular imprinted polymers for detection of low molecular mass analytes

Biomimetic recognition elements employed for the detection of analytes are commonly based on proteinaceous affibodies, immunoglobulins, single‐chain and single‐domain antibody fragments or aptamers. The alternative supra‐molecular approach using a molecularly imprinted polymer now has proven utility in numerous applications ranging from liquid chromatography to bioassays. Despite inherent advantages compared with biochemical/biological recognition (which include robustness, storage endurance and lower costs) there are few contributions that describe quantitative analytical applications of molecularly imprinted polymers for relevant small molecular mass compounds in real‐world samples. There is, however, significant literature describing the use of low‐power, portable piezoelectric transducers to detect analytes in environmental monitoring and other application areas. Here we review the combination of molecularly imprinted polymers as recognition elements with piezoelectric biosensors for quantitative detection of small molecules. Analytes are classified by type and sample matrix presentation and various molecularly imprinted polymer synthetic fabrication strategies are also reviewed.

Direct acoustic profiling of DNA hybridisation using HSV type 1 viral sequences

We describe the detection of specific, conserved DNA sequences of herpes simplex virus (HSV) type 1 by means of a novel, high sensitivity acoustic biosensor. Repeated assays on planar and polymeric carboxylic acid- and biotin-presenting surface chemistries enabled statistical comparison of assay specificity and sensitivity and evaluation of assay Z-factor scores. Using a three minute hybridisation with NeutrAvidin capture for signal enhancement, it was possible to detect HSV viral nucleic acids at 5.2 x 10(-11) M concentration.

A signal amplification assay for HSV type 1 viral DNA detection using nanoparticles and direct acoustic profiling

BackgroundNucleic acid based recognition of viral sequences can be used together with label-free biosensors to provide rapid, accurate confirmation of viral infection. To enhance detection sensitivity, gold nanoparticles can be employed with mass-sensitive acoustic biosensors (such as a quartz crystal microbalance) by either hybridising nanoparticle-oligonucleotide conjugates to complimentary surface-immobilised ssDNA probes on the sensor, or by using biotin-tagged target oligonucleotides bound to avidin-modified nanoparticles on the sensor. We have evaluated and refined these signal amplification assays for the detection from specific DNA sequences of Herpes Simplex Virus (HSV) type 1 and defined detection limits with a 16.5 MHz fundamental frequency thickness shear mode acoustic biosensor.ResultsIn the study the performance of semi-homogeneous and homogeneous assay formats (suited to rapid, single step tests) were evaluated utilising different diameter gold nanoparticles at varying DNA concentrations. Mathematical models were built to understand the effects of mass transport in the flow cell, the binding kinetics of targets to nanoparticles in solution, the packing geometries of targets on the nanoparticle, the packing of nanoparticles on the sensor surface and the effect of surface shear stiffness on the response of the acoustic sensor. This lead to the selection of optimised 15 nm nanoparticles that could be used with a 6 minute total assay time to achieve a limit of detection sensitivity of 5.2 × 10-12 M. Larger diameter nanoparticles gave poorer limits of detection than smaller particles. The limit of detection was three orders of magnitude lower than that observed using a hybridisation assay without nanoparticle signal amplification.ConclusionsAn analytical model was developed to determine optimal nanoparticle diameter, concentration and probe density, which allowed efficient and rapid optimisation of assay parameters. Numerical analysis and subsequent associated experimental data suggests that the response of the mass sensitive biosensor system used in conjunction with captured particles was affected by i) the coupled mass of the particle, ii) the proximal contact area between the particle and the sensor surface and iii) the available capture area on the particle and binding dynamics to this capture area. The latter two effects had more impact on the detection limit of the system than any potential enhancement due to added mass from a larger nanoparticle.