Development of a new biosensor array and lab-on-a-chip for portable applications using a label-free detection method

The detection and quantification of cardiac biomarkers in serum is crucial to diagnose patients in the early stage of a disease. The recent advances in microfluidics technology can improve diagnostics by reducing the application time and integrating several clinical analysis into a single, portable device called lab-on-a-chip (LOC). The development of such immunosensing LOC is a major thrust of the rapidly growing bionanotechnology industry. It involves a multidisciplinary research effort encompassing microfluidics, microelectronics and biochemistry. This thesis work focused on the development of immunoassays on microfabricated gold inter-digitated transducers (IDT) on silica and glass substrates. The concept of label-free, affinity-based biosensing is introduced with a special emphasis to impedance spectroscopy. Different protocols involving the covalent immobilization of cancer risk marker (human epidermal growth factor, hEGFR) and cardiac risk marker proteins C reactive protein (CRP), interleukin (IL6) and nicotinamide phosphoribosyltransferase (Nampt) single stranded deoxyribonucleic acid were investigated. For this, IDTs were fabricated using integrated circuit (IC) fabrication processes providing compatibility for the integration of electronic circuits, for single-chip and lab-on-a-chip biosensing applications. The thesis also involves development of a poly dimethylsiloxane (PDMS)-based fluidic system comprising on-chip actuated mechanism for multi-target immunosensing applications. The fluidic flow is controlled by an applied hydraulic pressure on the micropump. Label-free affinity type sensing was carried out using two different biological recognition elements (a) immunosensing approach using antibodies for hEGFR and IL-6 was employed and the function of the LOC was analyzed for detection of hEGFR and IL-6 as model analytes. A detection limit of 0.1ng/ml of hEGFR and IL-6 in serum was obtained without any signal amplification. (b) label-free affinity-based methodology using ssDNA aptamers specific for Nampt to develop an aptasensor and obtained a detection limit of 1 ng/ml in serum for Nampt, which is the most sensitive detection range with the application of the aptamer for Nampt

Label-free biosensors for the detection and quantification of cardiovascular risk markers

This paper presents a biosensor implementation for the detection of protein molecules using specific antibodies. Affinity sensors allow the detection and quantification of target molecules in complex mixtures by affinity-based interactions. Immobilized antibody molecules are the probes that bind to specific protein molecules (targets) in biological fluids. In this study, inter-digitated electrodes in the form of capacitance on glass slide were designed, fabricated and used to measure the changes in the dielectric properties of the inter-digitated capacitances. Our results in this study present that with a careful design of micro-interdigitated capacitors, a wider dynamic range and higher sensitivity can be achieved for the detection and quantification of C-Reeactive Protein

A new lab-on-chip transmitter for the detection of proteins using RNA aptamers

A new RNA aptamer based affinity biosensor for CReactive Protein (CRP), a risk marker for cardiovascular disease was developed using interdigitated capacitor (IDC), integrated in Voltage Controlled Oscillator (VCO) and output signal is amplified using Single Stage Power Amplifier (PA) for transmitting signal to receiver at Industrial, Scientific and Medical (ISM) band. The Lab-on-Chip transmitter design includes IDC, VCO and PA. The design was implemented in IHP 0.25μm SiGe BiCMOS process; post-CMOS process was utilized to increase the sensitivity of biosensor. The CRP was incubated between or on interdigitated electrodes and the changes in capacitance of IDC occurred. In blank measurements, the oscillation frequency was 2.464GHz whereas after RNA aptamers were immobilized on open aluminum areas of IDC and followed by binding reaction processed with 500pg/ml CRP solution, the capacitance shifted to 2.428GHz. Phase noise is changed from -114.3dBc/Hz to -116.5dBc/Hz

Sensitive detection of Nampt(PBEF/Visfatin)in human serum for point-of-care applications using aptamer based capacitive biosensor

NAMPT is a multifunctional protein, also known as visfatin or pre-B cell colony-enhancing factor, which exists as the rate-limiting intracellular enzyme for nicotinamide adenine dinucleotide (NAD) synthesis starting from nicotinamide [1]. The plasma Nampt levels are reported to have correlation with obesity and obese related metabolic disease, such as Type 2 diabetes mellitus (T2DM), cardiovascular diseases [2] and hyperlipidemia [3] due to association with lipoprotein and cholesterol. Therefore, sensitive detection of Nampt potentially enable accurate diagnosis of T2DM, cardiovascular and hyperlipidemia diseases. In this study, for the first time, we developed an ssDNA aptamer that specifically bind Nampt (Kd=72.52 nM) in human serum by systematic evolution of ligands by exponential enrichment (SELEX) process. Nampt-specific ssDNA aptamers were then applied as the recognition molecules for the development of a capacitive biosensor using non-Faradaic impedance spectroscopy (nFIES), which converts the biological binding event into a quantifiable signal for sensitive and efficient detection of the Nampt (Fig. 1). The interaction of aptamer-Nampt induced the change in dielectric properties, charge distribution, and conductivity. The limit of detection was 1 ng/ml with a dynamic range of upto 50 ng/ml in serum and this range is under the clinical requirements both in the normal Nampt levels, which is 15.8 ng/ml, and in the T2DM patients level, which is 31.9 ng/ml. This assay system for Nampt detection using aptamers is a potential alternative approach for applications in clinical studies and Point-Of-Care health technologies

Development of a new biosensor array and lab-on-a-chip for portable applications using a label-free detection method

The detection and quantification of cardiac biomarkers in serum is crucial to diagnose patients in the early stage of a disease. The recent advances in microfluidics technology can improve diagnostics by reducing the application time and integrating several clinical analysis into a single, portable device called lab-on-a-chip (LOC). The development of such immunosensing LOC is a major thrust of the rapidly growing bionanotechnology industry. It involves a multidisciplinary research effort encompassing microfluidics, microelectronics and biochemistry. This thesis work focused on the development of immunoassays on microfabricated gold inter-digitated transducers (IDT) on silica and glass substrates. The concept of label-free, affinity-based biosensing is introduced with a special emphasis to impedance spectroscopy. Different protocols involving the covalent immobilization of cancer risk marker (human epidermal growth factor, hEGFR) and cardiac risk marker proteins C reactive protein (CRP), interleukin (IL6) and nicotinamide phosphoribosyltransferase (Nampt) single stranded deoxyribonucleic acid were investigated. For this, IDTs were fabricated using integrated circuit (IC) fabrication processes providing compatibility for the integration of electronic circuits, for single-chip and lab-on-a-chip biosensing applications. The thesis also involves development of a poly dimethylsiloxane (PDMS)-based fluidic system comprising on-chip actuated mechanism for multi-target immunosensing applications. The fluidic flow is controlled by an applied hydraulic pressure on the micropump. Label-free affinity type sensing was carried out using two different biological recognition elements (a) immunosensing approach using antibodies for hEGFR and IL-6 was employed and the function of the LOC was analyzed for detection of hEGFR and IL-6 as model analytes. A detection limit of 0.1ng/ml of hEGFR and IL-6 in serum was obtained without any signal amplification. (b) label-free affinity-based methodology using ssDNA aptamers specific for Nampt to develop an aptasensor and obtained a detection limit of 1 ng/ml in serum for Nampt, which is the most sensitive detection range with the application of the aptamer for Nampt

A nanostructured-nickel based interdigitated capacitive transducer for biosensor applications

This paper presents, for the first time, the development of a nickel-based, reliable alternative electrode for biosensor applications. The electrodes are formed of nanosized nickel patterned as interdigitated structure to form a capacitive biosensor platform. To test the sensing performance of new biosensing platform, we investigated cardiovascular risk marker (C-reactive protein - CRP) and to check the specificity of the bioassay bovine serum albumin (BSA) and prostate specific antigen (PSA) were employed as controls. The nickel-electrode surface was covalently functionalized and characterized by Fourier transform infrared spectroscopy (FT-IR). The detection and quantification of CRP-antigen was performed by impediometric measurements. A detection model for the new biosensing platform was developed using the spectral impediometric responses and are matched with the specific antibody-antigen interaction-model, using the Cole-Cole model parameters. The results and analysis shown that the nanosized, nickel-based-electrode surface could be a good potential to be a cost effective alternative to the conventional gold electrodes with a comparable sensing performance, as shown for CRP, with a detection limit of 1-250 ng/ml. Furthermore, nano-sized nickel-based capacitive electrodes were fabricated using integrated circuit (IC) fabrication processes, providing compatibility for the integration of electronic circuits for single-chip and lab-on-chip biosensing applications

A novel magnetic particle-modified electrochemical sensor for immunosensor applications

In this study, a novel magnetic particle-modified capacitive sensor was reported for the detection of cancer markers. A gold interdigitated (GID) capacitor transducer was modified using magnetic beads (MB) for signal enhancement and the optimal frequency range and magnetic bead amount were determined. The platform was initially tested using C-reactive protein (CRP) as the model analyte and the methodology was then transferred for multiple marker detection with the aim of precise disease diagnostics. For the first time, the protein biomarkers of lung cancer including carcinoembryonic antigen (CEA), epidermal growth factor receptor (hEGFR) and cancer antigen 15-3 (CA15-3) were investigated with a capacitive sensor. The threshold levels of the markers to indicate the cancer are higher than 5 ng mL−1 (CEA), 64 ng mL−1 (hEGFR) and 50 U mL−1 (CA15-3), respectively. CEA and hEGFR could successfully be detected in the concentration range of 5 pg mL−1 to 1 ng mL−1 while CA15-3 was detected in the range of 1–200 U mL−1 with a high specificity. Our study demonstrates a highly specific capacitive immunoassay, presenting a potential alternative tool for early and precise diagnosis of cancer disease

Gold nanoparticle modified capacitive sensor platform for multiple marker detection

The detection and quantification of cancer biomarkers in human blood is crucial to diagnose patients in the early stage of a disease. The recent advances in biosensor technology can improve detection by reducing the application time and cost without an invasive approach. In this study, a highly sensitive, novel nanoparticle-modified capacitive sensor was developed for the detection of cancer markers. The current work mainly focused on developing a surface modification protocol for achieving higher sensitivity using Au-NPs. An interdigitated electrode (IDE) transducer was modified using gold nanoparticles (Au-NPs) for signal enhancement, the platform was initially optimized with a small size IL-6 protein and the methodology was then applied for multiple marker detection with the aim of precise disease diagnostics. Carcinoembryonic antigen (CEA) and epidermal growth factor receptor (hEGFR) could be successfully detected in the concentration range of 20–1000 pg mL−1 while cancer antigen 15-3 (CA15-3) was detected in the range of 10–200 U mL−1. These results show an increase of sensitivity by five-fold with respect to those not modified, demonstrating a highly sensitive and specific capacitive immunoassay that has a great potential for the use of early diagnosis of cancer disease

Label-free capacitive biosensor for the detection of a panel of disease biomarkers using microelectrode arrays

Disease biomarker panel that include C-reactive protein (CRP), TNF, IL-1 and IL-6 have strong and consistent relationships between markers of inflammation and future cardiovascular risk (CVR) events. Early detection of these biomarkers enables prevention of the prevailing disease risk. Here, we present label-free and sensitive detection of multiple biomarkers using interdigitated (IDA) capacitor arrays immobilized with specific antibodies. Two different lab-on-a-chip formats have been employed for multiple biomarker detection on arrays of IDA-capacitors; (I) each IDA-capacitor in an array was immobilized with a specific antibody and (II) each IDA-capacitor was immobilized with multiple antibodies in equimolar ratios in arrays (Fig. 1). Detection of single and multiple protein biomarkers on format I and II, respectively was performed by capacitive/dielectric constant measurements. Our initial results for detection of single biomarkers (format I) showed the detection range 25 ng/ml-25 pg/ml for CRP, 25-500 pg/ml for TNF and IL-6, and 1-25 ng/ml for IL-1. Further, detection of multiple biomarkers (format II) was carried out by immobilizing equimolar ratio’s of three types of antibodies (CRP, TNF, IL-6) and the resulting response to the mixture of biomarkers was lower in three orders compared to that found with IDA-capacitor immobilized with pure antibodies (format I) (Fig. 2A and B). The capacitive biosensor for panels of inflammation and CVR markers show significant clinical value and provide great potential for detection of protein markers in suspected subjects for early diagnosis

Label-free capacitive biosensor for sensitive detection of multiple biomarkers using gold interdigitated capacitor arrays

In this study, a highly sensitive and label-free multianalyte capacitive immunosensor was developed based on gold interdigitated electrodes (GID) capacitor arrays to detect a panel of disease biomarkers. C-reactive protein (CRP), TNF alpha, and IL6 have strong and consistent relationships between markers of inflammation and future cardiovascular risk (CVR) events. Early detection of a panel of biomarkers for a disease could enable accurate prediction of a disease risk. The detection of protein biomarkers was based on relative change in capacitive/dielectric properties. Two different lab-on-a-chip formats were employed for multiple biomarker detection on GID-capacitors. In format I, capacitor arrays were immobilized with pure forms of anti-CRP, -TNF alpha, and -IL6 antibodies in which each capacitor array contained a different immobilized antibody. Here, the CRP and IL6 were detected in the range 25 pg/ml to 25 ng/ml and 25 pg/ml to 1 ng/ml for TNFa in format I. Sensitive detection was achieved with chips co-immobilized (diluted) with equimolar mixtures of anti-CRP, -IL6, and -TNF alpha antibodies (format II) in which all capacitors in an array were identical and tested for biomarkers with sequential incubation. The resulting response to CRP, IL6, and TNF alpha in format II for all biomarkers was found to be within 25 pg/ml to 25 ng/ml range. The capacitive biosensor for panels of inflammation and CVR markers show significant clinical value and provide great potential for detection of biomarker panel in suspected subjects for early diagnosis