Versatile Biosensor Research Articles

Nanozyme Sensor Arrays for Detecting Versatile Analytes from Small Molecules to Proteins and Cells.

Nanozymes have emerged as promising alternatives to overcome the high cost and low stability of natural enzymes. Nanozymes with peroxidase-like activities have been studied to construct versatile biosensors by using specific biorecognition ligands (such as enzymes, antibodies, and aptamers) or molecularly imprinted polymers (MIPs). However, the use of bioligands compromises the high stability and low cost promise of nanozymes, while the MIPs may not be applicable to multiplex detection. To address these limitations, here we constructed the nanozyme sensor arrays based on peroxidase-like Pt, Ru, and Ir nanozymes. The cross-reactive nanozyme sensor arrays were successfully used for the detection of biothiols and proteins as well as the discrimination of cancer cells because of the differential nonspecific interactions between the components of the sensor arrays and the analytes. The usefulness of the nanozyme sensor arrays was further validated by the detection of blind unknown samples, where 28 of 30 biothiols and 42 of 45 proteins were correctly identified. Moreover, the practical application of the nanozyme sensor arrays was demonstrated by the successful discrimination of biothiols in serum and proteins in human urine.

GTP as a peroxidase-mimic to mediate enzymatic cascade reaction for alkaline phosphatase detection and alkaline phosphatase-linked immunoassay

Guanosine triphosphate (GTP), a substrate for RNA synthesis, plays a pivotal role in life-form metabolism at the cellular level. Herein, we first showed that GTP exhibited an intrinsic peroxidase-mimic activity, accelerating H2O2-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to produce a color reaction. Compared to GTP, weaker catalytic activity was observed from guanosine diphosphate (GDP) and virtually no catalytic activity was observed from guanosine monophosphate (GMP) and guanosine. Alkaline phosphatase (ALP) can catalyze the dephosphorylation of GTP with in situ generation of GDP, GMP and guanosine, which followed by receding the oxidation of TMB. Inspired by this, a novel colorimetric sensor for ALP detection was designed based on the enzymatic cascade reaction by coupling ALP-catalyzed dephosphorylation of GTP with GTP-catalyzed oxidation of TMB. This method allowed ALP sensing in a range from 0.01 to 100 U/L with a detection limit of 0.009 U/L, which was sensitive enough for ALP activity assay in biological samples (the normal ALP level range of adult serum is 20–140 U/L). Moreover, ALP is widely used in enzyme-linked immunosorbent assays (ELISA) as a signal reporter, the proof-of-concept ELISA for alpha-fetoprotein (AFP) detection was established. This unexpected discovery not only extends the new biological function of GTP, but also holds great promise for development of versatile biosensors that initiate the concentration changes of GTP.

Highly sensitive ratiometric electrochemical DNA biosensor based on homogeneous exonuclease III-assisted target recycling amplification and one-step triggered dual-signal output

A unique and versatile ratiometric electrochemical DNA biosensor was designed for the first time by integration of homogeneous exonuclease III-assisted target recycling amplification and one-step triggered dual-signal output. The presence of the target DNA would hybridize with the specifically designed ferrocene-labeled hairpin probe (Fc-P1) and initiate homogeneous exonuclease III-assisted target recycling amplification, which led to the decrease of the local concentration of Fc-P1. The target DNA dependent amount of remaining Fc-P1 could be conveniently monitored by hybridize the remaining Fc-P1 with the methylene blue-labeled hairpin probe ((MB-PP1) on the sensor electrode to trigger the MB tags away from while the Fc tags close to the sensing interface. Therefore, such recognition event can be translated into the homogeneous amplification coupled with one-step triggered dual-signal ratiometric electrochemical readout. The proposed biosensor achieves a detection limit of 12.8 fM within a linear range of 0.02 pM to 2 nM for target DNA species related to oral cancer over expressed 1, along with a favorable specificity. More importantly, the biosensor had been applied in detection of target DNA in artificial saliva sample and provided significant potential application in clinical analysis. This strategy possessed good capacity to integrate the nuclease amplification techniques with ratiometric output modes, and would open new opportunities for sensitive detection of other biorecognition events.

Two-dimensional molybdenum disulfide (MoS2) with gold nanoparticles for biosensing of explosives by optical spectroscopy

As one of the most important transition-metal dichalcogenides (TMDs) materials, molybdenum disulfide (MoS2) has gained extensive attention for its marvelous optoelectronic properties. In this study, nanocomposites of two-dimensional (2D) molybdenum disulfide (MoS2) with plasmonic noble metal nanoparticles were synthesized by a one-step green process. Under irradiation, 2D MoS2 in the nanocomposites absorbed photons and generated carriers in a wide spectrum. Then MoS2 coupled to gold nanoparticles and produced the enhanced electromagnetic field in the nanosturctures, leading to outstanding optical properties. In virtue of bio-compatibilities of the particles, the nanocomposites were chemically modified with specific peptides to construct an optical biosensor for explosive detection. When exposed to 2,4,6-trinitrotoluene (TNT), the biosensor showed significant absorption peak changes in visible spectra with a concentration-dependent behavior. The biosensor could detect TNT at the concentration as low as 2 × 10−7 M. For selectivity, the biosensor could differentiate TNT from other nitroaromatic explosives that have extremely similar structures to TNT, such as 4-nitrotoluene (4-NT) and 2,6-dinitrotoluene (DNT). All the results suggested that the biosensor based on the nanocomposites of 2D MoS2 coupling to plasmonic nanoparticles exhibited remarkable optical performance, which provided a promising approach to design versatile biosensors to detect biochemical molecules.

Peptide cleavage induced assembly enables highly sensitive electrochemiluminescence detection of protease activity

Proteases perform essential functions in a multitude of physiological processes and participate in many human diseases. Probing protease activity sensitively and accurately is critical for both basic research and clinical diagnosis. Herein, a novel optical biosensor for high-sensitive detection of protease was developed based on the specific protease cleavage of synthesized peptide substrate and subsequent assembly between exposed cysteine and Ru(bpy)32+-2-cyanobenzothiazole (Ru(bpy)32+-CBT). The assembly complexes can be enriched by streptavidin coated magnetic beads on work electrode for high-sensitive electrochemiluminescence (ECL) analysis. Using trypsase and caspase-3 as the examples, the limit of detection (LOD) of 8.4 × 10−4 U mL−1 and 5 × 10−6 U mL−1 were obtained respectively. This represents one of most sensitive protease assay reported. Current peptide cleavage induced assembly (PCIA) method can be easily extended to detect other proteases by changing peptide substrate sequence, shows its great potential as versatile biosensor in sensing techniques and clinical diagnosis.

A novel strategy for amplified probing versatile biomolecules through a photoswitchable biocatalytic cascade

We now present a novel signal amplification strategy of the photoswitchable enzyme cascade that can be used for versatile biosensing applications. Specifically, the biocatalysis of alkaline phosphatase (ALP) allows the formation of a nano-trigger of catechol coordinated TiO2 nanoparticles (NPs), which subsequently activates the activity of horseradish peroxidase (HRP) for oxidizing its typical substrate of 3,3’,5,5’-tetramethylbenzidine (TMB) under visible light irradiation. This photoswitchable enzyme cascade has powerful signal amplification effect, enabling us to construct advanced colorimetric sensing platform for probing ALP-based bioassays with ultra-high sensitivity. A low detection limit of 4 × 10−4 U/L was obtained for probing the activity of ALP. In addition, an immunoassay protocol to detect the cancer biomarker of human antigen α-fetoprotein (AFP, taken as a model) with the detection limit as low as 0.02 pg/mL also validated the versatility of the biocatalytic cascade as the amplified signaling transducer. Different from the conventional enzyme cascades in which the product of one enzyme is the substrate of the other, this study sets up the first example of nanomaterial mediated photoswitchable enzyme cascade, which may find wide applications in bioassays that relying on enzymatic catalysis for signal readout.

Photoinduced electron transfer from polymer-templated Ag nanoclusters to G-quadruplex-hemin complexes for the construction of versatile biosensors and logic gate applications.

In this paper, fluorescent Ag nanoclusters (Ag NCs) templated by hyperbranched polyethyleneimine (PEI) are utilized as a versatile probe through the photoinduced electron transfer (PET) between PEI-Ag NCs and G-quadruplex-hemin complexes. In the presence of hemin and target molecule, the specific conjugation with its aptamer induces the conformational change of the DNA sequence, releasing the G-quadruplex sequence part. Once the G-quadruplex-hemin complexes are introduced, electron transfer from the PEI-Ag NCs to G-quadruplex-hemin complexes occurs, resulting in fluorescence quenching. Through changing the sensing DNA sequence, this novel PET system enables the specific detection of target DNA and adenosine triphosphate (ATP) with the wide linear range of 1-200nM and 5-500nM, respectively, and the corresponding limit of detection as low as 0.3nM for target DNA and 1.5nM for ATP. In addition, the proposed method is successfully applied to the determination of ATP in human serum samples with satisfactory recoveries, and a logic gate is fabricated using target molecules and hemin as inputs and the fluorescence signal of PEI-Ag NCs as an output.

Nanoparticle-based fluoroionophore for analysis of potassium ion dynamics in 3D tissue models and in vivo.

The imaging of real-time fluxes of K+ ions in live cell with high dynamic range (5-150 mM) is of paramount importance for neuroscience and physiology of the gastrointestinal tract, kidney and other tissues. In particular, the research on high-performance deep-red fluorescent nanoparticle-based biosensors is highly anticipated. We found that BODIPY-based FI3 K+-sensitive fluoroionophore encapsulated in cationic polymer RL100 nanoparticles displays unusually strong efficiency in staining of broad spectrum of cell models, such as primary neurons and intestinal organoids. Using comparison of brightness, photostability and fluorescence lifetime imaging microscopy (FLIM) we confirmed that FI3 nanoparticles display distinctively superior intracellular staining compared to the free dye. We evaluated FI3 nanoparticles in real-time live cell imaging and found that it is highly useful for monitoring intra- and extracellular K+ dynamics in cultured neurons. Proof-of-concept in vivo brain imaging confirmed applicability of the biosensor for visualization of epileptic seizures. Collectively, this data makes fluoroionophore FI3 a versatile cross-platform fluorescent biosensor, broadly compatible with diverse experimental models and that crown ether-based polymer nanoparticles can provide a new venue for design of efficient fluorescent probes.

Enzyme Multilayers on Graphene-Based FETs for Biosensing Applications.

Electrochemical sensors represent a powerful tool for real-time measurement of a variety of analytes of much significance to different areas, ranging from clinical diagnostics to food technology. Point-of-care devices which can be used at patient bedside or for online monitoring of critical parameters are of great importance in clinical daily routine. In this work, portable, low-cost electrochemical sensors for a fast and reliable detection of the clinically relevant analyte urea have been developed. The intrinsic pH sensitivity of reduced graphene oxide (rGO)-based field-effect transistors (FETs) was exploited to monitor the enzymatic hydrolysis of urea. The functionalization of the sensor platform using the layer-by-layer technique is especially advantageous for the immobilization of the biorecognition element provided that this approach preserves the enzyme integrity as well as the rGO surface. The great selectivity of the enzyme (urease) combined with the high sensitivity of rGO-based FETs result in the construction of urea biosensors with a limit of detection (LOD) of 1μM and a linear range up to 1mM. Quantification of Cu2+ with a LOD down to 10nM was performed by taking advantage of the specific inhibition of urease in the presence of heavy metals. These versatile biosensors offer great possibilities for further development of highly sensitive enzyme-based FETs for real-time, label-free detection of a wide variety of clinically relevant analytes.

UV-light-induced synthesis of PEI-CuNCs based on Cu2+-quenched fluorescence turn-on assay for sensitive detection of biothiols, acetylcholinesterase activity and inhibitor

Polyethyleneimine-protected copper nanoclusters (PEI-CuNCs) were synthesized and applied to detect Cu2+, biothiols, and acetylcholinesterase (AChE). A facile UV-light-induced method was used in the synthesize process to obtain PEI-CuNCs with high stability and strong fluorescence intensity. Based on the quenching mechanism, a label-free assay for sensing Cu2+ by PEI-CuNCs was developed with the limit of detection (LOD) of 0.12 μM. Biothiols with SH functional groups could react with Cu2+ to form the RSH–Cu2+ complex, making Cu2+-triggered fluorescence quenching turned on. Two biothiols, glutathione and cysteine, were determined with LODs of 0.26 μM and 0.34 μM within linear ranges of 0.5–25 μM and 1–25 μM, respectively. AChE could hydrolyze acetylthiocholine into thiocholine (TCh), which reacted with Cu2+ to form TCh–Cu2+ complex and turned on Cu2+-quenched fluorescence. LOD of AChE was calculated to be 1.38 mU/mL within a linear range from 3 to 200 mU/mL. A typical inhibitor of AChE, tacrine, was also detected by this convenient fluorescence turn-on assay with the corresponding IC50 value (the inhibitor concentration required for 50% inhibition of the enzyme activity) of 53.4 nM. Moreover, the assay was successfully applied to the detection of biothiols and AChE activity in human serum samples. Results demonstrated the great potential of PEI-CuNCs and paved a new avenue for exploring their versatile biosensing applications.

ScFv-modified graphene-coated IDE-arrays for ‘label-free’ screening of cardiovascular disease biomarkers in physiological saline

Fatty-acid binding proteins (FABP) and myeloperoxidases (MPO) are associated with many chronic conditions in humans and considered to be important biomarkers for diagnosis of cardiac diseases. Here we assemble a new electrical biosensor platform based on graphene-coated interdigitated electrode arrays (IDE-arrays) towards ultrafast, label-free screening of heart type-FABP and MPO. Arrays of nanoscale (nanoIDE) and microscale (microIDE) electrode-arrays were fabricated on wafer-scale by combining nanoimprint and photolithography processes. Chemical vapor deposition grown multilayer graphene was transferred onto nano/microIDE-arrays and used as a high surface-to-volume ratio electrical transducer. Novel biofunctional layers of specially engineered anti-h-FABP and anti-MPO single-chain fragment variables (scFv) were immobilized onto graphene-coated IDE-array sensor platform for electrical detection of h-FABP and MPO in physiological saline. scFv fragments show increased sensitivity in comparison to the state-of-the-art competitive ELISA for their higher affinity towards target analytes. Deploying FABP and MPO specific scFvs as receptor molecules onto our high-sensitivity graphene-coated IDE-arrays with identical sensor characteristics and assays covering clinically relevant concentrations in physiological saline, we demonstrate realization of a simple and versatile biosensor platform capable of high performance cardiac-bioassays for point-of-care applications.

Low-cost, in-liquid measuring system using a novel compact oscillation circuit and quartz-crystal microbalances (QCMs) as a versatile biosensor platform

Abstract. Quartz-crystal microbalances (QCMs) are commercially available mass sensors which mainly consist of a quartz resonator that oscillates at a characteristic frequency, which shifts when mass changes due to surface binding of molecules. In addition to mass changes, the viscosity of gases or liquids in contact with the sensor also shifts the resonance but also influences the quality factor (Q-factor). Typical biosensor applications demand operation in liquid environments leading to viscous damping strongly lowering Q-factors. For obtaining reliable measurements in liquid environments, excellent resonator control and signal processing are essential but standard resonator circuits like the Pierce and Colpitts oscillator fail to establish stable resonances. Here we present a low-cost, compact and robust oscillator circuit comprising of state-of-the-art commercially available surface-mount technology components which stimulates the QCMs oscillation, while it also establishes a control loop regulating the applied voltage. Thereby an increased energy dissipation by strong viscous damping in liquid solutions can be compensated and oscillations are stabilized. The presented circuit is suitable to be used in compact biosensor systems using custom-made miniaturized QCMs in microfluidic environments. As a proof of concept we used this circuit in combination with a customized microfabricated QCM in a microfluidic environment to measure the concentration of C-reactive protein (CRP) in buffer (PBS) down to concentrations as low as 5 µg mL−1.

Design of Artificial Riboswitches as Biosensors.

RNA aptamers readily recognize small organic molecules, polypeptides, as well as other nucleic acids in a highly specific manner. Many such aptamers have evolved as parts of regulatory systems in nature. Experimental selection techniques such as SELEX have been very successful in finding artificial aptamers for a wide variety of natural and synthetic ligands. Changes in structure and/or stability of aptamers upon ligand binding can propagate through larger RNA constructs and cause specific structural changes at distal positions. In turn, these may affect transcription, translation, splicing, or binding events. The RNA secondary structure model realistically describes both thermodynamic and kinetic aspects of RNA structure formation and refolding at a single, consistent level of modelling. Thus, this framework allows studying the function of natural riboswitches in silico. Moreover, it enables rationally designing artificial switches, combining essentially arbitrary sensors with a broad choice of read-out systems. Eventually, this approach sets the stage for constructing versatile biosensors.

High sensitivity surface plasmon resonance biosensor for detection of microRNA and small molecule based on graphene oxide-gold nanoparticles composites

A versatile and sensitive surface plasmon resonance (SPR) biosensor based on two layers of graphene oxide-gold nanoparticles (GO-AuNPs) composites was designed for the detection of microRNA (miRNA) and small molecule adenosine. The bottom layer, which acted as a functionalized substrate on the sensor chip, provided a high specific surface area convenient for the immobilization of capture DNA molecules. The upper layer served as a signal-amplification element. By employing these two layers of GO-AuNPs composites, the dual amplification strategy was achieved so that a measurement of miRNA-141 with a detection limit of 0.1fM was obtained. Moreover, the developed SPR biosensor showed decent selectivity toward miRNA-200 family members. Especially, the SPR biosensor demonstrated its applicability for the detection of miRNA-141 in cancer cell extractions, and the results obtained were consistent with those obtained by qRT-PCR. Interestingly, small molecule adenosine could also be detected using this SPR biosensor in combination with a split aptamer. Considering the superior sensitivity, selectivity and generality, this work promised much potential for the detection of various biomolecules.

Exonuclease-Catalyzed Methylene Blue Releasing and Enriching onto a Dodecanethiol Monolayer for an Immobilization-Free and Highly Sensitive Electrochemical Nucleic Acid Biosensor.

Herein, a unique and versatile immobilization-free electrochemical nucleic acid biosensor architecture is proposed for the first time based on the catalyzed release of a methylene blue (MB)-tagged mononucleotide by exonuclease III (Exo III) and the successive enrichment onto a dodecanethiol monolayer, which can be attributed to the hydrophobic force between the alkyl chain of the dodecanethiol monolayer and the hydrophobic part of the MB-tagged mononucleotide. The fabricated biosensor demonstrates considerable advantages including assay simplicity, rapidness, and high sensitivity owing to its immobilization-free and homogenous operation for the biorecognition and amplification process. A low detection limit of approximately 1 pM toward the target DNA could be achieved with an excellent selectivity. The proposed immobilization-free electrochemical biosensing strategy was also extended for the assay of Exo I and III activity. Furthermore, it might be easily extended for the detection of a wide spectrum of targets and thus provide a promising avenue for the development of immobilization-free and sensitive electrochemical biosensors.

Rational Design of Electrochemical DNA Biosensors for Point‐of‐Care Applications

AbstractElectrochemical DNA biosensors show great potential for the sensitive and sequence‐specific detection of disease pathogens. To date, although there are a large number of published articles showing various sensing strategies of an electrochemical biosensor, only a few of those studies actually reach the commercialization phase, because addressing one technical issue would usually be at the expense of another. Moreover, there are various adoptable formats in electrochemical biosensors. Current approaches may or may not use enzymes, immobilize DNA probes, label the sensing substrates with an electroactive reporter molecule, or any combination of these. Although much has been paid to individual advantages and disadvantages, there are a very limited number Review articles studying and analyzing the synergistic aspects of various detection strategies when combined with each other. It is the aim of this Review to highlight the hotspots for innovation when these strategies are used in tandem, in order to create more streamlined improvements in making a versatile biosensor platform that can be administered at point of care.

Label-Free and Regenerative Electrochemical Microfluidic Biosensors for Continual Monitoring of Cell Secretomes.

Development of an efficient sensing platform capable of continual monitoring of biomarkers is needed to assess the functionality of the in vitro organoids and to evaluate their biological responses toward pharmaceutical compounds or chemical species over extended periods of time. Here, a novel label‐free microfluidic electrochemical (EC) biosensor with a unique built‐in on‐chip regeneration capability for continual measurement of cell‐secreted soluble biomarkers from an organoid culture in a fully automated manner without attenuating the sensor sensitivity is reported. The microfluidic EC biosensors are integrated with a human liver‐on‐a‐chip platform for continual monitoring of the metabolic activity of the organoids by measuring the levels of secreted biomarkers for up to 7 d, where the metabolic activity of the organoids is altered by a systemically applied drug. The variations in the biomarker levels are successfully measured by the microfluidic regenerative EC biosensors and agree well with cellular viability and enzyme‐linked immunosorbent assay analyses, validating the accuracy of the unique sensing platform. It is believed that this versatile and robust microfluidic EC biosensor that is capable of automated and continual detection of soluble biomarkers will find widespread use for long‐term monitoring of human organoids during drug toxicity studies or efficacy assessments of in vitro platforms.

Optimization and characterization of a flow cell for heat-transfer-based biosensing

In this article, we report on the development of a flow cell optimized for the heat-transfer method, a versatile biosensing technique. The design of the flow cell ensures that the heat flow is focused with minimal heat loss through the surroundings of the cell. This results in a more stable measuring signal and an improved sensitivity of the measuring technique. The sensor was characterized by performing background measurements in air, water, and phosphate buffered saline (PBS) solution. Heat flow through the setup was simulated using COMSOL in order to provide insight in the contribution of convection to the heat flow and recommendations for possible future improvements to the cell. Additionally, a two-step algorithm for calculating thermal resistance was defined, allowing the user to accurately derive thermal conductivity from experimental data. Finally, the potential of the flow cell for bacteria (Escherichia coli) detection was assessed and compared with the results obtained in the original HTM setup in a similar experiment. This experiment demonstrates that we were able to improve the limit-of-detection (LoD) to 2.10 × 104 colony forming units (CFU) mL−1 by changing the geometry of the measuring cell. Sensor setup for thermal biodetection experiments a directed heat flow.

Evolution of a split RNA polymerase as a versatile biosensor platform

Biosensors that transduce target chemical and biochemical inputs into genetic outputs are essential for bioengineering and synthetic biology. Current biosensor design strategies are often limited by a low signal-to-noise ratio, the extensive optimization required for each new input, and poor performance in mammalian cells. Here we report the development of a proximity-dependent split RNA polymerase (RNAP) as a general platform for biosensor engineering. After discovering that interactions between fused proteins modulate the assembly of a split T7 RNAP, we optimized the split RNAP components for protein-protein interaction detection by phage-assisted continuous evolution (PACE). We then applied the resulting activity-responsive RNAP (AR) system to create biosensors that can be activated by light and small molecules, demonstrating the 'plug-and-play' nature of the platform. Finally, we validated that ARs can interrogate multidimensional protein-protein interactions and trigger RNA nanostructure production, protein synthesis, and gene knockdown in mammalian systems, illustrating the versatility of ARs in synthetic biology applications.

Creation of Antigen-Dependent β-Lactamase Fusion Protein Tethered by Circularly Permuted Antibody Variable Domains.

Antibody-based molecular switches that are able to recognize a range of exogenous antigens can be highly useful as a versatile biosensor. However, regulating the catalytic activity of enzymes by antibodies is still hard to achieve. Here, we describe a design method of unique antibody variable region Fv introduced with two circular permutations, called Clampbody. By tethering the Clampbody to a circularly permuted TEM-1 β-lactamase (BLA), we successfully constructed a genetically encoded molecular switch Cbody-cpBLA that shows antigen-dependent catalytic activity.