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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 ( f p ) 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 ( f c ) ranges obtained from noise spectroscopy analysis can qualitatively differentiate between solution containing only AfB1, AfB1 and NSTs and no AfB1. However f c 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 f p and f c values in 11 categories each using k -means clustering algorithm and then applies a simple combinational digital logic on the clusters of f p s and f c s 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. [ABSTRACT FROM AUTHOR]

The use of porous silicon (PS) as an electrochemical biosensor has been reviewed. For electrochemical biosensing, the enhanced surface area is not the only advantage of using PS. In fact, depending on the transduction mechanisms, various issues like modulation of electric field lines through the pores and its interface with the biomolecules and ions of the analytes, nature of PS–metal contacts, orientation of the molecules within the pores and modification of the effective spring constant of the structure leading to a change in the resonant frequency have significant impact on sensitivity, reproducibility and longevity. In this review, we provide a critical assessment of the various factors which are actually responsible for enhanced performance of PS electrochemical biosensors. Formation procedures of PS for electrochemical biosensing have been mentioned. The mechanism of surface modification and derivatization has been briefly discussed. The contact formation techniques have been outlined. Four different categories of electrochemical biosensors using PS have been analysed in details with emphasis on equivalent circuit models and the specific role of PS in the electrical transduction mechanisms. Finally the existing challenges and the future prospects of such devices for commercial use are highlighted. [ABSTRACT FROM AUTHOR]

We report an electrochemically fabricated silicon oxide nanoporous structure for ultrasensitive detection of AfB1 in food by shift in peak frequency corresponding to maximum sensitivity. It has been observed that the impedance sensitivity changes from 19% to 40% (which is only twice) where as the peak frequency shifts from 500 Hz to 50 kHz, for a change in concentration from 1 fg/ml to 1 pg/ml. This has been attributed to the combined effect of the significant pore narrowing with increasing AfB1 concentration and the opposing nature of impedance change within the nanopores and the conducting substrate immediately below the nanoporous layer. [ABSTRACT FROM AUTHOR]

Abstract: Microimpedance affinity biosensors using interdigitated electrodes are being extensively employed for label free and rapid detection of bacteria molecules. To improve the sensitivity of such sensors, it is required to maximize the antibody immobilization and bacteria captured for an applied input concentration through proper quantification and also optimize the electrode geometry to achieve maximum signal to noise ratio. In this paper, a biomolecule compatible electrical model has been developed which quantifies the number of immobilized antibody and captured bacteria by simple calculations from impedance spectroscopy results which are comparable to the accuracy obtained by conventional time consuming and sometimes expensive biochemical methods and can also estimate the electrode impedance associated with each biomolecule. The model has been applied for the detection of Escherichia coli K12 bacteria by antibody–antigen binding method on gold electrodes with PBS as the background solution. The quantitative estimation of the actual number of antibody immobilized and bacteria captured for input concentration of 103 and 106 CFU/ml on the gold surface in this case has been matched with the conventional optical density methods and radioactive labeling method respectively. A quantitative relationship between the geometrical parameters and the output signal has been deduced after extracting the electrode impedance for each biomolecule and the effects of transducer geometry on the sensitivity of this biosensor have been matched with recently reported experimental results for the same system. Thus the proposed model enables quantitative estimation of the antibody immobilized and bacteria captured and quantitative prediction of the optimum geometry for maximum sensitivity eliminating the need for trial and error experimentation. This will lead to a rapid and cost effective method for performance enhancement of microimpedance biosensors with different topologies. [Copyright &y& Elsevier]

Abstract: 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 1–2μm 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 103 CFU/ml in pure culture using a 3mm×1mm 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Oxidized porous silicon (PS) is a common topographical biocompatible substrate that potentially provides a distinct in vitro environment for better understanding of in vivo behavior. But in the reported studies on oxidized PS, cell-cell and cell-substrate interactions have been detected only by fluorescent labeling. This paper is the first attempt to investigate real-time sensing of these interactions on HaCaT cells by label-free impedance spectroscopy on oxidized PS of two pore diameters (50 and 500 nm). One of the major requirements for successful impedance spectroscopy measurement is to restrict the channeling of electric field lines through the pores. To satisfy this criterion, we have designed the pore depths after analyzing the penetration of the medium by using computational fluid dynamics simulation. A distributed electrical model was also developed for estimating the various cellular attributes by considering a pseudorandom distribution of pores. It is observed from the impedance measurements and from the model that the proliferation rate increases for 50 nm pores but decreases for 500 nm pores compared to that for planar substrates. The rate of decrease in cell substrate separation (h) in the initial stage is more than the rate of increase in cell-cell junction resistance (Rb) corresponding to the initial adhesion phase of cells. It is observed that Rb and h are higher for 50 nm pores than those for planar substrates, corresponding to the fact that substrates more conducive toward cell adhesion encourage cell-cell interactions than direct cell-substrate interactions. Thus, the impedance spectroscopy coupled with the proposed theoretical framework for PS substrates can sense and quantify the cellular interactions. [ABSTRACT FROM AUTHOR]

Diverse efforts undertaken to mitigate the Debye screening effect of field-effect transistor (FET) biosensors have achieved higher sensitivity. However, the charge transfer or induction mechanism between the captured biomolecules and the sensing surface, resulting in shift of surface potential and, hence, the drain current magnitude, shows poor selectivity for real time detection of ultra-low biomolecule concentration in unamplified samples. Herein, enabling direct interaction of the target molecule with edge passivated thermally reduced graphene oxide, through the template of molecularly imprinted polymer (MIP), specific trap induced characteristic bulges in the noise spectrum has been observed only in the presence of target molecule. Such a phenomenon has not been observed either with antibodies or aptamers as receptors. Hence, the direct interface of MIP with a graphene FET (GFET) results in the generation of an additional electronic label in FET transduction. As a proof of concept, this principle has been validated for serotonin detection in serum in the presence of other neurotransmitters, resulting in detection limit of 0.05 fM in real time, which is more than two orders of magnitude lower than existing reports and, hence, is expected to pave the way for advancing GFET based sensors toward ultrasensitive point-of-care applications without requiring complex machine learning algorithms. [ABSTRACT FROM AUTHOR]

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 ( C q ) 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 C q and C EDL changes in opposite direction after AFB1 capture and the nature of frequency dependence of C q and C EDL 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. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • Attomolar detection of HBsAg in serum using ZnO nanorods based liquid gated Bio-FET with vertical electrode configuration. • Enhanced penetration of electric field lines across nanorods elevates sensitivity value than usual lateral electrode pattern. • A change in threshold voltage and carrier mobility, leads to a sensitivity around 66% at 1 fM and a detection limit of 20 aM. [ABSTRACT FROM AUTHOR]

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 ( f p ) from the steady state sensitivity characteristics and the first cut off frequency ( f c ) 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 0.04 fM–1 pM and with an accuracy of around 90% beyond 1 pM and within 25 nM in blood serum. This is the most sensitive report on multiple virus quantification using label free method. [ABSTRACT FROM AUTHOR]

The authors would like to make corrections to [1, Fig. 7(b) and (c)] due to some post-processing carried out in these figures. After capturing the SEM images—top view [Fig. 7(b)] and cross section [Fig. 7(c)] in [1], we started measuring the pore dimensions with image processing software. We wanted to measure both the lateral and the vertical diameters of the pores in Fig. 7(b) (since they are not exactly circular). For this purpose, a boundary was constructed around some of the pores with textured lines but mistakenly these lines were not removed during transferring the pictures to the manuscript, which is responsible for the distortion in [1, Fig. 7(b)]. In Fig. 7(c) also, we wanted to measure the pore length, for which textured lines were drawn along the length of the pore, leading to some changes in the color contrast at the mouth of the pore from the original SEM picture. These lines also should have been removed from [1, Fig. 7(c)]. [ABSTRACT FROM AUTHOR]

Abstract: In this paper, optimization of the protocol for covalent antibody immobilization on macroporous silicon solid supports of various porosities, which is recently being employed as a promising substrate for biosensors, has been reported. Covalent binding of antibody has been carried out by silanization and crosslinker attachment on the substrate. For maximum antibody immobilization on macroporous silicon, all the individual processes have been separately optimized for the first time in terms of treatment time, pH, concentration, incubation time and others with the help of optical density measurements. The optimum treatment of the surface after every step has been further reconfirmed by detailed EDX analysis, SEM measurements and contact angle measurements. It has been observed that the density of antibody binding increases with increasing porosity and for a 70% porosity macroporous sample it is almost three times more than that of planar silicon which is significantly higher than the previous comparative reports on planar silicon and macroporous silicon. The amount of properly oriented HIgG antibodies has been estimated by quantification of the alkaline phosphate conjugated protein A binding by optical density measurements. [Copyright &y& Elsevier]

Abstract: Macroporous silicon is a promising material for wide spectrum of electrical applications ranging from photonic devices, microsystems engineering, supercapacitors to biosensors. Fabrication of good quality ohmic contact on macroporous silicon is a prerequisite for its widespread use in electrical sensor applications but it is also challenging since shallow nanopores are always present within the macropores. This paper aims at the formation of low resistance and fairly ohmic contacts on p-type macroporous silicon with 1.5μm pore diameter. Electrical contacts formed by vacuum evaporation of aluminium exhibit non-ohmic behaviour owing to the presence of the shallow nanoporous region formed on the surface of crystallites in macroporous silicon. To overcome this problem a controlled pre-annealing step at a temperature of 900°C for 30min leading to closure of shallow nanopores through coalescence of nanocrystallites of silicon due to grain growth has been carried out. The growth parameters are computed from the FESEM characteristics of the macroporous silicon preannealed at 900°C for varying time. For optimally preannealed macroporous silicon the I–V characteristics of macroporous silicon has been found to exhibit fairly ohmic behaviour with vacuum evaporated aluminium. Detailed electrical characterization of vacuum evaporated contacts on both unannealed and preannealed macroporous silicon has been carried out using modified TLM technique. It has been found that the specific contact resistance is almost five times lower for the latter. [Copyright &y& Elsevier]

• The impact of surface defects has been explored in terms of sensitivity. • Tuning of surface roughness and sensing of PSA have been done. • Transconductance of the sensors degrade monotonically with increasing defectdensity. • The optimized sensor has a PSA detection limit of 0.06 fM. • Nonoptimized sensor with initial detection limit of 0.1 fM has longevity of 4 months. In this work, the impact of surface defects in zinc oxide (ZnO) thin films on the performance of field effect transistor (FET) biosensors has been explored. Here, ZnO thin films have been fabricated using electron beam evaporation with various deposition parameters to tune the surface roughness and defect state density. The films have been deployed for sensing prostate specific antigen (PSA) after anti-PSA antibody immobilization. Interestingly, it has been observed that though the transconductance of the sensors degrade monotonically with increasing defect density, the antibody binding density has a non-monotonic behaviour due to enhanced steric hindrance effect beyond a certain limit of surface roughness. There is a variation of sensitivity by 15 times with different physical properties of ZnO and the optimized sensor has a PSA detection limit of 0.06 fM. Further, the sensor with the best sensitivity retains its performance upto 2 months while a non-optimized sensor with initial PSA detection limit of 0.1 fM has longevity of 4 months. Thus, it can be envisaged that tuning the surface defect state in ZnO FET biosensor is an essential requirement not only to ensure a low detection limit and high sensitivity but also to assure a reliable long term performance. [ABSTRACT FROM AUTHOR]