Deformed graphene FET biosensor on textured glass coupled with dielectrophoretic trapping for ultrasensitive detection of GFAP.

Numerous efforts have been undertaken to mitigate the Debye screening effect of FET biosensors for achieving higher sensitivity. There are few reports that show sub-femtomolar detection of biomolecules by FET mechanisms but they either suffer from significant background noise or lack robust control. In this aspect, deformed/crumpled graphene has been recently deployed by other researchers for various biomolecule detection like DNA, COVID-19 spike proteins and immunity markers like IL-6 at sub-femtomolar levels. However, the chemical vapor deposition (CVD) approach for graphene fabrication suffers from various surface contamination while the transfer process induces structural defects. In this paper, an alternative fabrication methodology has been proposed where glass substrate has been initially texturized by wet chemical etching through the sacrificial layer of synthesized silver nanoparticles, obtained by annealing of thin silver films leading to solid state dewetting. Graphene has been subsequently deposited by thermal reduction technique from graphene oxide solution. The resulting deformed graphene structure exhibits higher sensor response towards glial fibrillary acidic protein (GFAP) detection with respect to flat graphene owing to the combined effect of reduced Debye screening and higher surface area for receptor immobilization. Additionally, another interesting aspect of the reported work lies in the biomolecule capture by dielectrophoretic (DEP) transport on the crests of the convex surfaces of graphene in a coplanar gated topology structure which has resulted in 10 aM and 28 aM detection limits of GFAP in buffer and undiluted plasma respectively, within 15 min of application of analyte. The detection limit in buffer is almost four decades lower than that documented for GFAP using biosensors which is is expected to pave way for advancing graphene FET based sensors towards ultrasensitive point-of-care diagnosis of GFAP, a biomarker for traumatic brain injury.

Experiment and FEM Analysis of Silica Nanoparticle-Based Impedance Immunosensor for Sensitivity Enhancement.

This article investigates the impact of incorporating silica nanoparticles of varying diameters in label free impedance immunosensor. It has been observed that even if the surface area improvement has been adjusted to be similar for all the diameters, the sensitivity is enhanced by five times at a particular diameter of 100 nm due to the optimum combination of intersection with electric field lines and surface convexity. This study has enabled the detection of 0.1 fM Hep-B surface antigen with a reliable sensitivity of around 75%. Further, it has been observed that the SNR corresponding to 0.1 fM is 20 dB only for 100 nm particle. This SNR is comparable to a recent report on Hep-B virus detection but the limit of detection in the proposed sensor is lowered by more than three orders of magnitude.

Liquid gated ZnO nanorod FET sensor for ultrasensitive detection of Hepatitis B surface antigen with vertical electrode configuration.

Detection of the Hepatitis-B surface antigen at the attomolar level is demonstrated using antibody functionalized liquid gated ZnO nanorods field effect transistor (FET) biosensor with vertical electrode configuration. The sensor is operated in heterodyne mode at high frequency to overcome the problem of Debye screening effect in physiological analyte. Enhanced penetration of the electric field lines through the nanorods enables significant improvement in the limit of detection and sensitivity compared to that of the conventional lateral electrode configuration. The combined effect of the probable change in the threshold voltage and the carrier mobility for vertical electrode configuration lead to a sensitivity of around 75% at 1 fM (which is an enhancement by 200%) and a detection limit of 20 aM with a dynamic range from 20 aM to 1 pM. The detection limit which is achieved with the proposed label free sensor in physiological analyte using antibodies is lowered by more than three orders of magnitude compared to the existing reports.

Label-Free Biomolecule Detection in Physiological Solutions With Enhanced Sensitivity Using Graphene Nanogrids FET Biosensor.

Recently, graphene nanogrid sensor has been reported to be capable of sub-femtomolar sensing of Hepatitis B (Hep-B) surface antigen in buffer. However, for such low concentration of Hep-B in serum, it has been observed during real-time operation that there is an overlap of around 50% in the drain-source current sensitivity values between different concentrations of the target biomolecule, in the range from 0.1 to 100 fM. This has been attributed to the fact that the concentration of non-specific antigen in serum being significantly higher than that of the target antigen, there is a considerable deviation in the number of captured target antigen for the same concentration. Further, this degree of overlap varies from one set to another set of sensor, depending on the statistical variations in the sensor fabrication process. This phenomenon challenges the quantification of target antigen for ultralow limit in physiological analyte. In this paper, we introduce probabilistic neural network (PNN) for quantification of Hep-B down to 0.1 fM in serum using graphene nanogrids field-effect transistor biosensor. The sensor has been operated in heterodyne mode in the frequency range of 100 kHz to 1 MHz applied between drain and source to overcome the problem of Debye screening effect. The application of PNN limits the quantification error within 10% in the range of 0.1 to 100 fM in contrast to 77% and 66% using polynomial fit and static neural network models, respectively. Further, the proposed methodology lowers the detection limit of Hep-B in serum by more than three orders of magnitude compared with the state-of-the-art, real-time, label-free sensors.

Quantitative differentiation of multiple virus in blood using nanoporous silicon oxide immunosensor and artificial neural network.

In spite of the rapid developments in various nanosensor technologies, it still remains challenging to realize a reliable ultrasensitive electrical biosensing platform which will be able to detect multiple viruses in blood simultaneously with a fairly high reproducibility without using secondary labels. In this paper, we have reported quantitative differentiation of Hep-B and Hep-C viruses in blood using nanoporous silicon oxide immunosensor array and artificial neural network (ANN). The peak frequency output (fp) from the steady state sensitivity characteristics and the first cut off frequency (fc) from the transient characteristics have been considered as inputs to the multilayer ANN. Implementation of several classifier blocks in the ANN architecture and coupling them with both the sensor chips, functionalized with Hep-B and Hep-C antibodies have enabled the quantification of the viruses with an accuracy of around 95% in the range of 0.04fM-1pM and with an accuracy of around 90% beyond 1pM and within 25nM in blood serum. This is the most sensitive report on multiple virus quantification using label free method.

A graphene field effect capacitive Immunosensor for sub-femtomolar food toxin detection.

In this paper we report the sensing of aflatoxin B1(AFB1) by field effect capacitive method using electrophoretically deposited reduced graphene oxide (RGO) films for the first time. The RGO film has been characterized using SEM, surface profilometer and Raman spectroscopy. It has been observed that both quantum capacitance of RGO (Cq) and effective electrical double layer capacitance (C(EDL)) contribute significantly towards the overall sensitivity for molar concentration in the range of 20-50 mM. As Cq and CEDL changes in opposite direction after AFB1 capture and the nature of frequency dependence of Cq and CEDL are different, the sensitivity shows a minima at a particular frequency. Interestingly, the sensitivity minima is also dependent on AFB1 concentration. Further, the maximum sensitivity obtained is around 30% for 10(-4) ppt (0.1 fg/ml) AFB1 which is greater than 1.5 times that of previous reports. This has been possible through the enhanced biomolecule immobilization capability of RGO. Thus the RGO based field effect capacitive sensor provides a combined advantage of both a high sensitivity and concentration dependent frequency behavior.

Noise spectroscopy as an efficient tool for impedance based sub-femtomolar toxin detection in complex mixture using nanoporous silicon oxide.

In this paper we demonstrate an efficient and non-interfering computational method for sub-femtomolar food toxin detection in complex mixture based on nanoporous silicon oxide impedance immunosensor by employing noise spectroscopy analysis at the peak frequency. It has been observed that the peak frequency (fp) values obtained from steady state impedance measurements cannot distinguish between solution with only the specific toxin, which is aflatoxin B1 (AfB1) and mixture of AfB1 with other non-specific toxins (NSTs), thus leading to erroneous quantification of AfB1 in complex mixture. On the other hand, the first cut-off frequency (fc) ranges obtained from noise spectroscopy analysis can qualitatively differentiate between solution containing only AfB1, AfB1 and NSTs and no AfB1. However fc values being very close for different concentration of AfB1 in pure solution and being overlapping for different mixtures cannot quantify AfB1 either in pure solution or in complex mixture. To address this problem, the proposed computational method first clusters the fp and fc values in 11 categories each using k-means clustering algorithm and then applies a simple combinational digital logic on the clusters of fps and fcs to obtain the final output, realizable with standard NAND-NOR gates. The output digital word differs only with AfB1 concentration and not with concentration of NSTs and is found to be capable of detecting sub-femtomolar AfB1 range down to 0.1 fg/ml not only in pure solution but also in complex mixture with as high as 1000 ng/ml NSTs. This is the most sensitive and selective report so far on electrochemical food toxin immunosensors.

Extended electrical model for impedance characterization of cultured HeLa cells in non-confluent state using ECIS electrodes.

Electric cell substrate impedance sensing has been widely used as a label free quantitative platform to study various cell biological processes and it is extremely essential to extract the parameters like the variation of the cell substrate spacing, changing projected area of the cell on the electrode and approximate cluster size during the non-confluent state to understand the mechanism of proliferation of the cells. The distributed analytical models developed so far to extract these parameters are applicable only under the conditions when the cells have become confluent. There are some lumped electrical models which have been reported for the non-confluent state but they do not provide correct estimate of the changing cell substrate spacing and the cell cluster size during growth. In this paper we develop extended distributed electrical models to characterize the impedance spectroscopy behavior of cultured HeLa cells in 200 Hz to 1 MHz range using eight well ECIS electrodes in the non-confluent state. The distributed model introduces some pseudo regularity in the arrangement of the non-confluent cells to extract the average ensemble of the significant parameters. The parameters extracted from the distributed model after 10 hours, 20 hours, and 30 hours of HeLa cell growth have been compared with the lumped circuit model and has been observed to fit the experimental data with a seven times improved fit quality factor. Further, the changing cell radius and cluster radius extracted at three different instants of time from the distributed analytical model have been found to match closely the microscopic observation in contrast to the lumped circuit model.

Microtrap electrode devices for single cell trapping and impedance measurement.

This paper reports the design and fabrication of electrode microtraps for single cell trapping and impedance measurement. In this work, the microtrap electrodes of parallel and elliptical geometry have been fabricated by electroplating of gold electrodes of optimum thickness. This has enabled the formation of electrode traps without requiring any precision alignment between separate insulating traps like PDMS and the bottom gold electrodes. Further the improved uniformity of the electric field between the trapping electrodes as observed from COVENTORWARE simulation significantly reduces the effect of cell position inside the microwell on the electrical measurement unlike previous reports. This makes it possible to directly extract the equivalent cell parameters from the electrical measurement without introducing any correction factor corresponding to cell position. We have performed impedance spectroscopy with both the microwell electrode structures with single HeLa cell at two different positions of trapping. It has been observed that there is almost no change in the extracted values of cell resistance and capacitance for different positions within parallel electrodes and there is only 0.7 % and 0.85 % change in cell resistance and capacitance for the two positions within elliptical electrodes. Thus these microwell electrode structures can be used as an improved and a more convenient platform for single cell electrical characterization.

Macroporous silicon based simple and efficient trapping platform for electrical detection of Salmonella typhimurium pathogens.

A thermally oxidized macroporous silicon substrate with simple electrode structure without interdigitated electrode configuration has been reported for the detection of Salmonella typhimurium pathogens by electrical impedance measurement using antibody-antigen binding method. Macroporous silicon which has been fabricated by anodizing silicon in HF and DMF solution is a regular network of pores of 1-2 microm diameters. This has been thermally oxidized to yield the surface hydrophilic for antibody immobilization as well as provide suitable electrical insulation of the metal contacts from the underlying conducting silicon substrate. The macroporous silicon surface has been immobilized by Salmonella specific antibody and has been tested with different concentration of S. typhimurium pathogens in phosphate buffer solution (PBS). It has been found that such macroporous silicon substrates is capable of detecting down to 10(3)CFU/ml in pure culture using a 3 mm x 1 mm electrode structure with a wide spacing of 1mm. The selectivity of the macroporous silicon substrates with reference to S. typhimurium has been tested to be satisfactory by carrying out controlled experiments with Escherichia coli O157:H7.