Biological Binding Events Research Articles

Surfactant-assisted synthesis of water-stable cobalt-imidazole metal organic frameworks as signal probes for chemiluminescent immunoassay

Application of metal-organic frameworks (MOFs) as signal probes for immunoassay has been greatly hampered by their chemical instability in aqueous solutions. To reduce the attack of water molecules and improve the water stability of MOFs, cetyltrimethyl ammonium bromide (CTAB) was used as a capping reagent to form a hydrophobic protective layer on the surface of MOFs. The Co-MOFs synthesized with CTAB-assisted approach (CTAB-Co-MOFs) can keep stable in water for no less than 45 days, while the stationary duration of those without CTAB assistance is only 3 days. The CTAB-Co-MOFs can vastly catalyze the reaction between luminol and H2O2, and generate very intensive chemiluminescent (CL) emission. The results of mechanism study show that CTAB-Co-MOFs are capable of promoting the generation of reactive oxygen species. Attributing to the improved water stability and outstanding catalytic activity of CTAB-Co-MOFs, a competitive immunoassay method was established for detecting acetamiprid by adopting them as CL probes. It shows a linear range of 0.01–25 ng mL-1 and a detection limit of 4.77 pg mL-1 (3σ). Successful detection of herbal medicines spiked with acetamiprid validated the accuracy of the CTAB-Co-MOFs-based immunoassay method. This work implies the possibility of using water-stable CTAB-Co-MOFs for tracing biological binding events.

Highly Sensitive Chemiluminescent Immunoassay of Mycotoxins Using ZIF-8-Derived Yolk-Shell Co Single-Atom Site Catalysts as Superior Fenton-like Probes.

Superior to traditional nanoscale catalysts, single-atom site catalysts (SASCs) show such merits as maximal catalysis efficiency and outstanding catalytic activity for the construction of analytical methodological platforms. Hereby, an in situ etching strategy was designed to prepare yolk-shell Co SASCs derived from ZIF-8@SiO2 nanoparticles. On the basis of direct chemical interactions between precursors and supports, the Co element with isolated atomic dispersion was anchored on ZIF-8@SiO2 nanoparticles. The Co SASCs possess high Fenton-like activity and thus can catalyze the decomposition of H2O2 to produce massive superoxide radical anions instead of singlet oxygen and hydroxyl radicals. With the activity for producing superoxide radical anion, Co SASCs can greatly improve the chemiluminescent (CL) response of a luminol system by 3133.7 times. Furthermore, the SASCs with active sites of Co-O5 moieties were utilized as the CL probes for establishment of an immunoassay method for sensitive detection of mycotoxins by adopting aflatoxin B1 as a mode analyte. The quantitation range is 10-1000 pg/mL, and the limit of detection is 0.44 pg/mL (3σ) for aflatoxin B1. The proof-of-principle work elucidates the practicability of direct chemical interactions between precursors and supports for forming SASCs with ultrahigh CL response, which can be extended to the exploitation of more sorts of SASCs for tracing biological binding events.

Passive Impedance Sensing Using a SAW Resonator-Coupled Biosensor for Zero-Power Wearable Applications

A bio-sensing scheme, which acquires impedance information of a capacitive biosensor by using the reflected RF signal from a surface acoustic wave (SAW) resonator connected to the biosensor, is proposed. This technique requires no power to be supplied to the biosensor node and hence is highly applicable to wearable applications. Theoretical analysis has demonstrated that the sensitivity of the SAW resonator-coupled biosensor is higher than that of traditional impedance loaded SAW sensors and therefore it is more suitable for measuring the very small impedance changes in biosensors. The passive detection of the change in the impedance of a capacitive biosensor, as a result of biological binding events associated with the capture of a target analyte, has been demonstrated by preliminary experimentation. Dry tests of the SAW coupled capacitive biosensor using a cable connected network analyzer showed the aF level capacitance measurement resolution, which was only achieved in transistor level circuits previously, could be attained. When liquid samples with concentrations of C-Reactive Protein (CRP) in the range of 0.1 to 2 μg/ml were applied to the biosensor, a corresponding change in the resonant frequency of the SAW resonator-coupled biosensor (in the order of sub-hundred kHz) was observed. This has demonstrated the potential for applying this technique in applications where a zero-power requirement at the biosensor node could be a distinct advantage, when the cable link between the network analyzer and the biosensor node is replaced by the RF transmission.

Plug-in label-free optical fiber DNA hybridization sensor based on C-type fiber Vernier effect

A reflective Fabry-Perot interference (FPI) label-free biosensor based on Vernier effect is designed and implemented for in situ real-time DNA hybridization detection. The sensor is made by splicing single-mode fiber (SMF), a segment of C-type fiber and SMF. The Vernier effect occurs between the solid cavity FPI and the air cavity FPI. In our experiment, a high sensitivity of 10791.12 nm/RIU is obtained in the refractive index (RI) range of 1.3311–1.3450. On this basis, we immobilized probe DNA (pDNA) on the surface of SMF to detect the complementary DNA (cDNA), demonstrating use for label-free sensing of DNA hybridization. The experimental results show that the sensor can detect 1 µM cDNA with high specificity. The paper demonstrates the first research of optical fiber DNA biosensor based on the open microcavity FPI Vernier effect. In addition, the sensor has the advantages of high sensitivity, strong stability, simple structure and simple fabrication. It can also be applied to other biomolecule detection based on probe-target biological binding events, which has great application potential in medical diagnostics, food science, genetic engineering and other biological fields.

Metal Nanoparticles and Carbon-Based Nanomaterials for Improved Performances of Electrochemical (Bio)Sensors with Biomedical Applications.

Monitoring human health for early detection of disease conditions or health disorders is of major clinical importance for maintaining a healthy life. Sensors are small devices employed for qualitative and quantitative determination of various analytes by monitoring their properties using a certain transduction method. A “real-time” biosensor includes a biological recognition receptor (such as an antibody, enzyme, nucleic acid or whole cell) and a transducer to convert the biological binding event to a detectable signal, which is read out indicating both the presence and concentration of the analyte molecule. A wide range of specific analytes with biomedical significance at ultralow concentration can be sensitively detected. In nano(bio)sensors, nanoparticles (NPs) are incorporated into the (bio)sensor design by attachment to the suitably modified platforms. For this purpose, metal nanoparticles have many advantageous properties making them useful in the transducer component of the (bio)sensors. Gold, silver and platinum NPs have been the most popular ones, each form of these metallic NPs exhibiting special surface and interface features, which significantly improve the biocompatibility and transduction of the (bio)sensor compared to the same process in the absence of these NPs. This comprehensive review is focused on the main types of NPs used for electrochemical (bio)sensors design, especially screen-printed electrodes, with their specific medical application due to their improved analytical performances and miniaturized form. Other advantages such as supporting real-time decision and rapid manipulation are pointed out. A special attention is paid to carbon-based nanomaterials (especially carbon nanotubes and graphene), used by themselves or decorated with metal nanoparticles, with excellent features such as high surface area, excellent conductivity, effective catalytic properties and biocompatibility, which confer to these hybrid nanocomposites a wide biomedical applicability.

In Situ Temperature-Compensated DNA Hybridization Detection Using a Dual-Channel Optical Fiber Sensor

A multifunction, high-sensitivity, and temperature-compensated optical fiber DNA hybridization sensor combining surface plasmon resonance (SPR) and Mach-Zehnder interference (MZI) has been designed and implemented. We demonstrate, for the first time to our knowledge, the dual-parameter measurement of temperature and refractive index (RI) by simultaneously using SPR and MZI in a simple single-mode fiber (SMF)-no-core fiber (NCF)-SMF structure. The experimental results show RI sensitivities of 930 and 1899 nm/RIU and temperature sensitivities of 0.4 and -1.4 nm/°C for the MZI and SPR, respectively. We demonstrate a sensitivity matrix used to simultaneously detect both parameters, solving the problem of temperature interference of RI variation-based biosensors. In addition, the sensor can also distinguish biological binding events by detecting the localized RI changes at the fiber's surface. We realize label-free sensing of DNA hybridization detection by immobilizing probe DNA (pDNA) onto the fiber as the probe to capture complementary DNA (cDNA). The experimental results show that the sensor can qualitatively detect cDNA after temperature compensation, and the limit of detection (LOD) of the sensor reaches 80 nM. The proposed sensor has advantages of high sensitivity, real time, low cost, temperature compensation, and low detection limit and is suitable for in situ monitoring, high-precision sensing of DNA molecules, and other related fields, such as gene diagnosis, kinship judgment, environmental monitoring, and so on.

Monitoring “Real-Time” Biological Binding Events on a Microelectrode Array

Because microelectrode arrays contain collections of spatially isolated electrodes that can each be individually addressed, they provide an intriguing platform for monitoring the activity of small molecules, peptides, and proteins. The arrays are utilized by placing the members of a molecular library on the surface of the array above the electrodes such that each unique member of the library is located on top of a unique electrode or a set of electrodes in the library. In this way, the binding activity on the molecular library and the electrochemical reactions happen on the electrodes are coupled. The binding events between the molecules on the surface of the array and molecules in solution can thus be monitored with the change of behavior of the electrochemical reactions on the electrodes. The overall strategy is illustrated in Figure below.We are expanding the types of experiments that can be employed to examine enzyme-peptide binding with the use of microelectrode array. We have previously shown RGD-peptide/ integrin, v-107/ VEGF, and R6A - Gαi1 interactions can be monitored on an array. While success of the synthetic chemistry can be ensured by fluorescence labeling, the binding can be monitored by cyclic voltammogram.Now, our focus has moved from distinguishing specific binding/non-specific binding/no binding to distinguishing different types of specific binding events. We are working to employ those electrochemical methods in conjunction with the traditional FRET studies in order to provide unique insights into the mechanism of peptide cleavage reactions. In so doing, we hope to add new capabilities to existing surface analytical methods. Figure 1

Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions.

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membraneinteractions, and viral budding, among other vital cellular processes. For efficient biological activity, cell membranes should be rigid enough to maintain the integrity of the cell and its compartments yet fluid enough to allow membrane components, such as proteins and functional domains, to diffuse and interact. This delicate balance of elastic and fluid membrane properties, and their impact on biological function, necessitate a better understanding of collective membrane dynamics over mesoscopic length and time scales of key biological processes, e.g., membrane deformations and protein binding events. Among the techniques that can effectively probe this dynamic range is neutron spin echo (NSE) spectroscopy. Combined with deuterium labeling, NSE can be used to directly access bending and thickness fluctuations as well as mesoscopic dynamics of select membrane features. This paper provides a brief description of the NSE technique and outlines the procedures for performing NSE experiments on liposomal membranes, including details of sample preparation and deuteration schemes, along with instructions for data collection and reduction. The paper also introduces data analysis methods used to extract key membrane parameters, such as the bending rigidity modulus, area compressibility modulus, and in-plane viscosity. To illustrate the biological importance of NSE studies, select examples of membrane phenomena probed by NSE are discussed, namely, the effect of additives on membrane bending rigidity, the impact of domain formation on membrane fluctuations, and the dynamic signature of membrane-protein interactions.

Biosensing on the Centrifugal Microfluidic Lab-on-a-Disc Platform

Lab-on-a-Disc (LoaD) biosensors are increasingly a promising solution for many biosensing applications. In the search for a perfect match between point-of-care (PoC) microfluidic devices and biosensors, the LoaD platform has the potential to be reliable, sensitive, low-cost, and easy-to-use. The present global pandemic draws attention to the importance of rapid sample-to-answer PoC devices for minimising manual intervention and sample manipulation, thus increasing the safety of the health professional while minimising the chances of sample contamination. A biosensor is defined by its ability to measure an analyte by converting a biological binding event to tangible analytical data. With evolving manufacturing processes for both LoaDs and biosensors, it is becoming more feasible to embed biosensors within the platform and/or to pair the microfluidic cartridges with low-cost detection systems. This review considers the basics of the centrifugal microfluidics and describes recent developments in common biosensing methods and novel technologies for fluidic control and automation. Finally, an overview of current devices on the market is provided. This review will guide scientists who want to initiate research in LoaD PoC devices as well as providing valuable reference material to researchers active in the field.

High-sensitivity Sagnac-interferometer biosensor based on exposed core microstructured optical fiber

A novel, high sensitivity Sagnac-interferometer biosensor based on exposed core microstructured optical fiber (ECF) has been designed and implemented in this paper. The exposed core fiber has noncircular symmetry and thus exhibits birefringence and can form a sensing element within a Sagnac loop interferometer. The exposed-core fiber design provides direct access to the evanescent field, allowing the measurement of bulk refractive index (RI) with a sensitivity of up to −3137 nm/RIU while maintaining the fiber’s robustness. The sensor can also detect the localized refractive index changes at the fiber core’s surface as the result of a biological binding event. We demonstrate the use of this sensor for label-free sensing of biological molecules by immobilizing biotin onto the fiber core as the probe to capture the target molecule streptavidin.

Label-Free and Recalibrated Multilayer MoS2 Biosensor for Point-of-Care Diagnostics.

Molybdenum disulfide (MoS2) field-effect transistor (FET)-based biosensors have attracted significant attention as promising candidates for highly sensitive, label-free biomolecule detection devices. In this paper, toward practical applications of biosensors, we demonstrate reliable and quantitative detection of a prostate cancer biomarker using the MoS2 FET biosensor in a nonaqueous environment by reducing nonspecific molecular binding events and realizing uniform chemisorption of anti-PSA onto the MoS2 surface. A systematic and statistical study on the capability of the proposed device is presented, and the biological binding events are directly confirmed and characterized through intensive structural and electrical analysis. Our proposed biosensor can reliably detect various PSA concentrations with a limit of 100 fg/mL. Moreover, rigorous theoretical simulations provide a comprehensive understanding of the operating mechanism of the MoS2 FET biosensors, and further suggests the enhancement of the sensitivity through engineering device design parameters.

Optical Monitoring of the Capillary Filling Dynamics Variation in Nanoporous Anodic Alumina toward Sensing Applications.

Fluid imbibition-coupled laser interferometry (FICLI) is a technique in which the kinetics of a fluid infiltrating a nanoporous anodic alumina (NAA) membrane is monitored by the interference of a laser beam at the membrane top and bottom surfaces. Further processing of the measured data results in an estimate of the pore radius. In this work, we study the accuracy of FICLI in the detection of small changes in pore radius, and we evaluate the possibility of using such detection as a sensing paradigm. The accuracy is estimated by measuring samples with increasing pore radius, obtained by successive wet etching steps, and repeatability is evaluated by using different liquids. For decreasing pore radius, samples obtained by the successive deposition of polyelectrolyte double layers are used. With the aim of evaluating the possibility of the FICLI method to sense biological binding events, BSA attachment detection is demonstrated by applying FICLI to samples before and after immobilization of the protein. Results show that the technique permits an accurate estimation of the pore radius, the pore-etching rate (with a radius variation of retch,DI = 1.05 nm/min ± 0.11 nm/min), and the polyelectrolyte double layer thickness (with a radius variation of rPAH/PSS = 3.2 nm ± 0.2 nm per polyelectrolyte double layer). Furthermore, the pore radius reduction measured after BSA immobilization (dBSA = 4.9 nm ± 1.1 nm) is in good agreement with the protein size, as reported in the literature. With these results, we provide a sound basis for the applicability of FICLI as a sensitive technique for the characterization of NAA pore radius modifications.

Highly Sensitive Colorimetric Cancer Cell Detection Based on Dual Signal Amplification.

Facile and efficient detection of cancer cells at their preclinical stages is one of the central challenges in cancer diagnostics. A direct, rapid, highly sensitive and specific biosensor for detection of cancer biomarkers is desirable in early diagnosis and prognosis of cancer. In this work, we developed, for the first time, an easy and intuitive dispersion-dominated colorimetric strategy for cancer cell detection based on combining multi-DNA released from an aptamer scaffold with cyclic enzymatic amplification, which was triggered by aptamer DNA conformational switch and demonstrated by non-cross-linking gold nanoparticles (Au NPs) aggregation. First, five kinds of messenger DNAs (mDNAs) were aligned on the cancer cell aptamers modified on magnetic beads (MBs) to form mDNAs-Apt-MBs biocompatible nanosensors. In the presence of target cells, the aptamer would bind to the receptors on the cell membranes, and mDNAs would be released, resulting in the first amplification that one biological binding event would cause the release of multiple kinds of mDNAs simultaneously. After magnetic separation, the released mDNAs were introduced into the cyclic enzymatic amplification to cleave more single strand DNA (ssDNA) fragments. Instead of modification of Au NPs, these fragments and mDNAs could be adsorbed on the surface of Au NPs to prevent particle aggregation and ensure the stability and color of solution in high salt environments. The linear response for HL-60 cells in a concentration range from 10 to 10(4) cells was obtained with a detection limit of four cells in buffer solution. Moreover, the feasibility of the proposed strategy was demonstrated in a diluted serum sample. This dual signal amplification method can be extended to other types of cancer cells, which has potential application in point-of-care cancer diagnosis.

Multivalent Glycopolymer-Coated Gold Nanoparticles.

Glycosylated noble metal nanoparticles are a useful tool for probing biological binding events due to their aggregation-induced color changes, particularly for lectins that have multiple binding sites. To overcome the challenges of colloidal instability, which leads to false-positive results, it is essential to add polymeric coatings to these particles. Here we describe a versatile, and reliable, approach to enable coating of gold nanoparticles using well-defined polymers, with carbohydrate end groups. This produces multivalent nanoparticles that are both colloidally stable, but still retain their rapid colorimetric responses to lectin binding.

Interferometric-type optical biosensor based on exposed core microstructured optical fiber

This work presents a novel biosensor using the multimode interference effect in an exposed core microstructured optical fiber (ECF). In this work biotin molecules are immobilized onto the ECF core surface to serve as the capturing probe for streptavidin, the target molecules. Since each distinct guided mode in the ECF interacts with the surrounding medium differently, the interference between any two specific modes will experience a fringe shift (or phase change) upon a change in the refractive index (RI) of the surrounding medium, or a localized RI change on the surface of the ECF core as a result of a biological binding event. In our experiment, the interferometric sensing platform was realized by splicing a section of ECF with lead-in and lead-out single mode fibers (SMFs). An interference pattern is obtained in the transmission spectrum as the result of multiple excited modes (excited and re-collected at the lead-in and lead-out splicing points) propagating in the ECF with different propagation constants. The interference pattern is non-uniform, indicating that there are more than two modes involved. Fast Fourier transform (FFT) is used to separate individual interference patterns that contribute to this complex spectrum and monitor their phase changes upon RI variation of the surrounding medium. In this way multiple RI sensitivities can be realized because each spatial frequency possesses a distinct sensitivity with respect to the surrounding RI. The operation of this device was validated by measuring the phase changes that occur when the sensing platform was subjected to solutions of different RIs or functionalized with different molecules. A biosensor was demonstrated based on this novel platform using biotin as the capturing probe to specifically detect streptavidin with low non-specific adsorption. The proposed platform is reliable, cost-effective, and offers a potential label-free biosensing alternative to the widely used surface plasmon resonance (SPR) technique.

Surface Functionalization of Ion-Sensitive Floating-Gate Field-Effect Transistors With Organic Electronics

Electrically conducting polymers are advantageous hybrid materials for microelectronic biosensors due to their high bandgap sensitivity, possibilities for nanoscale surface area formation, and well-developed surface bioconjugation strategies. In this paper, we investigated whether those organic conductors can also be used to functionalize ion-sensitive floating-gate field-effect transistors (ISFGFETs) designed to measure biological binding events. We first subjected our device to 100% relative humidity (RH) and proved its viability in such a humid environment. Subsequently, we drop-casted viscoelastic polyaniline emeraldine salt on pristine transistors to construct organo-functionalized devices. The modified ISFGFETs were stable in aqueous environments and sensitive to cationic polyethyleneimine. The directions of the ISFGFET threshold voltage (VT) shifts agree with the corresponding open-circuit potential variations for the same reaction and pH-sensitive behaviors of Al <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> sensing layer on the transistor. Such organo-modified ISFGFET sensor arrays are promising alternatives to traditional conductive polymer-based potentiometric biosensors due to their signal amplification, high throughput, and scalability advantages.

An efficient signal-on aptamer-based biosensor for adenosine triphosphate detection using graphene oxide both as an electrochemical and electrochemiluminescence signal indicator.

An efficient aptasensor was developed in which graphene oxide (GO) was employed as an indicator for both electrochemical impedance spectroscopy and electrochemiluminescence (ECL) signal generation. The aptasensor was fabricated by self-assembling the ECL probe of a thiolated adenosine triphosphate binding aptamer (ABA) tagged with a Ru complex (Ru(bpy)3(2+) derivatives) onto the surface of gold nanoparticle (AuNP) modified glassy carbon electrode (GCE). ABA immobilized onto AuNP modified GCE could strongly adsorb GO due to the strong π-π interaction between ABA and graphene oxide; ECL quenching of the Ru complex then takes place because of energy transfer and electron transfer, and a large increase of the electron transfer resistance (Ret) of the electrode. While in the presence of target adenosine triphosphate (ATP), the ABA prefers to form ABA-ATP bioaffinity complexes, which have weak affinity to graphene oxide and keep the graphene oxide away from the electrode surface, thus allowing the ECL signal enhancement, and in conjunction with the decrease of the Ret. Because of the high ECL quenching efficiency, unique structure, and electronic properties of graphene oxide, the Ret and ECL intensity versus the logarithm of ATP concentration was linear in the wide range from 10 pM to 10 nM with an ultra-low detection limit of 6.7 pM to 4.8 pM, respectively. The proposed aptasensor exhibited excellent reproducibility, stability, and outstanding selectivity, and ATP could be effectively distinguished from its analogues. More significantly, this efficient ECL aptasensor strategy based on GO acting both as an electrochemical and ECL signal indicator is general and can be easily extended to other biological binding events.

Conformational analysis of proteins with a dual polarisation silicon microring

Optical microresonator biosensors have proven to be a valid tool to perform affinity analysis of a biological binding event. However, when these microresonators are excited with a single optical mode they can not distinguish between a thin dense layer of biomolecules or a thick sparse layer. This means the sensor is "blind" to changes in shape of bound biomolecules. We succeeded in exciting a Silicon-on-Insulator (SOI) microring with TE and TM polarisations simultaneously by using an asymmetrical directional coupler and as such were able to separately determine the thickness and the density (or refractive index) of a bound biolayer. A proof-of-concept is given by determining both parameters of deposited dielectric layers and by analysing the conformational changes of Bovine Serum Albumin (BSA) proteins due to a change in pH of the buffer.

Electrochemiluminescence Ratiometry: A New Approach to DNA Biosensing

Inspired by dual-wavelength fluorescence ratiometric method which could reduce the influence from the environmental change, here, we present a novel dual-potential electrochemiluminescence (ECL) ratiometric sensing approach. CdS nanocrystal (NC) and luminol as two different ECL emitters are employed. ECL from CdS NCs coated on glassy carbon electrode at -1.25 V (vs SCE) could be quenched by closely contacted Pt nanoparticles (NPs) via a biological binding event, while ECL from luminol at +0.45 V (vs SCE) could be enhanced by the same Pt NPs, in the presence of their common coreactant of H₂O₂. Thus, the quenching of ECL from CdS NCs and the enhancement of ECL from luminol could indicate the same biological binding event. With the mp53 oncogene as a model DNA molecule, a molecular beacon (MB) containing a 20-base loop, which is complementary with the mp53 oncogene, is immobilized on CdS NCs/GCE first; Pt NPs are then captured on CdS NCs surface by DNA hybridization between the MB and mp53 oncogene labeled on Pt NPs. By measuring the ratio of ECL intensities at two excitation potentials, this approach could sensitively detect the concentration of target DNA in a wide range from 5.0 fM to 1.0 pM. The sensing scheme is general and can be utilized for many other biological binding events.

Highly Sensitive Reduced Graphene Oxide Impedance Sensor Harnessing π-Stacking Interaction Mediated Direct Deposition of Protein Probes

Graphene-based electrochemical impedance sensors have recently received much attention due to their outstanding sensing capability and economic viability. In this study, we present a novel means of constructing an impedance sensing platform via harnessing intrinsic π-stacking interactions between probe protein molecules and reduced graphene oxide (RGO) substrate, obviating the need for introducing external chemical groups often required for covalent anchoring of the probes. To achieve this goal, protein molecules used as a probe were denatured to render their hydrophobic residues exposed in order to facilitate their direct π-stacking interactions with the surface of RGO nanosheets. The protein molecules in denatured form, which would otherwise have difficulty in undergoing π-stacking interactions with the RGO surface, were found to uniformly cover the RGO nanosheets at high density, conducive to providing a graphene-based impedance sensing platform capable of detecting a probe-specific analyte at high sensitivity. The proof-of-concept performance of thus-constructed RGO-based impedance sensors was demonstrated via selective detection of biological binding events of antigen-antibody reaction at a femtomolar range. Notably, since the π-stacking interaction can occur on the entire RGO surface, it can desirably exclude a backfill process indispensable for the conventional biosensors to suppress background noise signals. Since the procedure of π-stacking mediated direct deposition of on-purpose denatured protein probes onto the RGO surface is facile and straightforward, the proposed strategy is anticipated to extend its applicability for fabrication of high performance graphene-based bio or chemical sensors.