Molecular Biosensors Research Articles

On the Potential for Tuning the Longitudinal Plasmon Band of a Distribution of Gold Nanorods Using a Tunable Laser

Gold nanorods hold great potential for bio sensing, imaging and therapy applications. The seed-mediated approach is the most widely used technique for the synthesis of gold nanorods. However, batches of nanorods synthesized using this method have a broadened longitudinal plasmon (LP) peak and uncertainty in the wavelength of the peak. This paper describes a technique to tune the synthesized nanorod distributions by using laser pulses at specific wavelengths that either reduce the width of the population LP peak or control its position. The overlap between the LP peaks of pairs of batches of gold nanorods was successfully reduced by at least 9%. Batches of nanorods with a sharper LP peak and/or a more desirable LP resonance wavelength could eventually be utilized in molecular biosensing and imaging applications requiring the simultaneous detection and differentiation of multiple aspectratio gold nanorods.

Construction of Electrodes Modified with Biopolymers for Biomolecular Sensing

Construction of Electrodes Modified with Biopolymers for Biomolecular Sensing

Engineering a Localized Surface Plasmon Resonance Platform for Molecular Biosensing

In this work, we introduce a new perspective on the development of Localized Surface Plasmon Resonance (LSPR) optical biosensors. Computational simulations, focused on the assessment of the LSPR spectrum and spatial distribution of the electromagnetic field enhancement near a metallic nanoparticle, elucidated the behavior of crucial parameters, as figure of merit, bulk and molecular sensitivity, which governs a LSPR sensor performance. Gold and silver nanospheres were explored as starting point to assess plasmonic optical characteristics of the nanostructured sensor platform. Here, for the first time in the literature, Campbell’s model was evaluated exploiting a NP size-dependence approach. The theoretical analyses indicate a nonlinear behavior of the bulk and molecular sensitivity as function of the NP size. Substantial LSPR peak shifts due to the adsorption of molecules layer on a NP surface were observed for nanoparticles with ~5 nm and ~40 nm radius. Moreover, on molecular sensing, LSPR peak shift is also determined by the thickness of adsorbed molecular shell layers. We observed that for 40 nm radius gold and silver nanospheres, significant LSPR peak shift could be induced by small (few nm) thickness change of the adsorbate shell layer. Moreover, this work provides insights on the LSPR behavior due to adsorption of molecular layer on a NP surface, establishing a new paradigm on engineering LSPR biosensor. Furthermore, the proposed approach can be extended to engineer an efficiently use of different nanostructures on molecular sensing.

Рамановское рассеяние света высокого спектрального разрешения в метфоpмине

AbstractHigh-resolution spectra of molecular vibrations in metformin, one of the most widely used antidiabetic drugs, which also possesses antiaging and antitumor properties, have been obtained by a high-sensitivity Raman-scattering technique. It is established that rather narrow (4–10 cm^–1) spectral lines observed in a 300–1200 cm^–1 range can be used for separating spectral components corresponding to the vibrations of particular functional groups. This circumstance makes metformin an interesting model for studying molecular biosensors.

Pre-Clinical Tests of an Integrated CMOS Biomolecular Sensor for Cardiac Diseases Diagnosis.

Coronary artery disease and its related complications pose great threats to human health. In this work, we aim to clinically evaluate a CMOS field-effect biomolecular sensor for cardiac biomarkers, cardiac-specific troponin-I (cTnI), N-terminal prohormone brain natriuretic peptide (NT-proBNP), and interleukin-6 (IL-6). The CMOS biosensor is implemented via a standard commercialized 0.35 μm CMOS process. To validate the sensing characteristics, in buffer conditions, the developed CMOS biosensor has identified the detection limits of IL-6, cTnI, and NT-proBNP as being 45 pM, 32 pM, and 32 pM, respectively. In clinical serum conditions, furthermore, the developed CMOS biosensor performs a good correlation with an enzyme-linked immuno-sorbent assay (ELISA) obtained from a hospital central laboratory. Based on this work, the CMOS field-effect biosensor poses good potential for accomplishing the needs of a point-of-care testing (POCT) system for heart disease diagnosis.

Molecular Biosensors for Electrochemical Detection of Infectious Pathogens in Liquid Biopsies: Current Trends and Challenges.

Rapid and reliable diagnosis of infectious diseases caused by pathogens, and timely initiation of appropriate treatment are critical determinants to promote optimal clinical outcomes and general public health. Conventional in vitro diagnostics for infectious diseases are time-consuming and require centralized laboratories, experienced personnel and bulky equipment. Recent advances in electrochemical affinity biosensors have demonstrated to surpass conventional standards in regards to time, simplicity, accuracy and cost in this field. The tremendous potential offered by electrochemical affinity biosensors to detect on-site infectious pathogens at clinically relevant levels in scarcely treated body fluids is clearly stated in this review. The development and application of selected examples using different specific receptors, assay formats and electrochemical approaches focusing on the determination of specific circulating biomarkers of different molecular (genetic, regulatory and functional) levels associated with bacterial and viral pathogens are critically discussed. Existing challenges still to be addressed and future directions in this rapidly advancing and highly interesting field are also briefly pointed out.

Ultra-fast and sensitive silicon nanobelt field-effect transistor for high-throughput screening of alpha-fetoprotein

In this paper, we described a novel fabrication technique for Silicon nanobelt field-effect transistors (SiNB FETs) based on the Local Oxidation of Silicon (LOCOS) process widely utilized in the manufacturing of microelectronics components and explore it to screened alpha-fetoprotein (AFP). LOCOS process is used as it enables thorough compatibility with complementary metal oxide semiconductor (CMOS) technology. Thus, the need for expensive lithography tools to define the nanoscale pattern is avoided. The SiNB FETs emerges as a powerful biosensor for ultrasensitive, label-free for biological/chemical detection and direct electrical readout. The analytical sensing results of the fabricated SiNB FETs employed as a biomolecular sensor for the early, real-time, and label-free screening of AFP can be understood in term of the change in charge density at the silicon nanobelt surface after functionalization. It is observed that, by tuning the gate voltage, the electrical linearity response of the system towards AFP extends its concentration range from 3ng/mL to 100ng/mL, allowing the screening of hepatocellular carcinoma (HCC). These results indicate that the detection of AFP under our direct, label-free, and ultrasensitive biosensor in a microfluidic channel could be one of the promising state-of-the-art techniques applicable as an AFP detector in real samples.

Three-Dimensionally Coupled THz Octagrams as Isotropic Metamaterials

Split-ring resonator (SRR) based metamaterials have been studied for the development of highly sensitive, small-sized, low-power chemical and biomolecular sensors. However, the anisotropic behavior arising from their two-dimensional (2D) structure presents substantial challenges leading to ambiguity in their transmission spectra. In this paper, we present the design of a three-dimensional (3D) isotropic octagram split-ring resonator (OSRR) demonstrating a three-dimensionally coupled resonance behavior that overcomes the anisotropic response of conventional 2D SRRs, leading to a strong, distortion-free, and polarization-invariant transmission response. The OSRR undergoes 3D coupling through the splits at the corners of the 3D structure (cube), which remains invariant under any polarization along the coordinate axes. The strong coupling between resonant segments provides the OSRR with 25 times higher sensitivity than the corresponding 2D structure, allowing the resonant frequency to be reliably monitored fo...

Aptamer-based F0F1-ATPase biosensor for Salmonella typhimurium detection

Abstract A novel biosensor was developed by conjugating Salmonella typhimurium aptamer with the “rotator” e-subunit of F0F1–ATPase within chromatophores through an anti-e-subunit antibody–biotin–avidin–biotin–aptamer linker to capture bacteria. The bacterial load decreased the rotation rate of F0F1-ATPase, which led to the decrease in ATP synthesis. The detection of S. typhimurium was based on proton flux change driven by the F0F1-ATPase-mediated ATP-synthesis, which was indicated by fluorescence probe, monitored by a fluorescence spectrometer. Under optimal conditions, the correlation between the relative fluorescence signal intensity and the concentration of S. typhimurium was observed to be linear within the range of 101 to 104 cfu·mL−1 (R2 = 0.9944). The limit of detection for S. typhimurium was as low as 10 cfu·mL−1. The results demonstrate that the F0F1-ATPase molecular motor biosensor can specifically detect S. typhimurium at low concentration, and will be a convenient, quick, and promising tool for detecting pathogens.

Fully Integrated Fluorescence Biosensors On-Chip Employing Multi-Functional Nanoplasmonic Optical Structures in CMOS

Integrated optical system-on-chip in silicon operating in the visible range can have a tremendous impact on enabling new applications in sensing and imaging through the ultra-miniaturization of complex optical instrumentation. CMOS technology has allowed an integration of optical detection circuitry for image sensors with massively large number of pixels. In this paper, we focus on techniques to realize complex optical-field processing elements inside CMOS by exploiting optical interaction with sub-wavelength metal nanostructures realized in the electrical interconnects layers, whose feature sizes are now in the sub-100-nm range. In particular, we present a fully integrated fluorescence-based bio-molecular sensor in 65-nm CMOS with integrated nanoplasmonic waveguide-based filters capable of more than 50 dB of rejection ratio across a wide range of incident angles. Co-designed with the integrated photo-detection circuitry, capacitive TIAs, and correlated double-sampling circuitry, the sensor is capable of detecting 48 zeptomoles of quantum dots on the surface with 52 fA of photodetector current with a fluorescence/excitation ratio of nearly −62 dB without any post-fabrication, external optical filters, lenses, or collimators. The ability to integrate complex nanoplasmonic metal structures with unique optical properties in CMOS with no post-processing creates the opportunity to enable large multiplexed assays on a single chip and a wide variety of applications, from in vitro to in vivo .

Single-cell profiling of dynamic cytokine secretion and the phenotype of immune cells.

Natural killer (NK) cells are a highly heterogeneous population of innate lymphocytes that constitute our first line of defense against several types of tumors and microbial infections. Understanding the heterogeneity of these lymphocytes requires the ability to integrate their underlying phenotype with dynamic functional behaviors. We have developed and validated a single-cell methodology that integrates cellular phenotyping and dynamic cytokine secretion based on nanowell arrays and bead-based molecular biosensors. We demonstrate the robust passivation of the polydimethylsiloxane (PDMS)-based nanowells arrays with polyethylene glycol (PEG) and validated our assay by comparison to enzyme-linked immunospot (ELISPOT) assays. We used numerical simulations to optimize the molecular density of antibodies on the surface of the beads as a function of the capture efficiency of cytokines within an open-well system. Analysis of hundreds of individual human peripheral blood NK cells profiled ex vivo revealed that CD56dimCD16+ NK cells are immediate secretors of interferon gamma (IFN-γ) upon activation by phorbol 12-myristate 13-acetate (PMA) and ionomycin (< 3 h), and that there was no evidence of cooperation between NK cells leading to either synergistic activation or faster IFN-γ secretion. Furthermore, we observed that both the amount and rate of IFN-γ secretion from individual NK cells were donor-dependent. Collectively, these results establish our methodology as an investigational tool for combining phenotyping and real-time protein secretion of individual cells in a high-throughput manner.

Engineering Intracellularly Retained Gaussia Luciferase Reporters for Improved Biosensing and Molecular Imaging Applications.

Gaussia luciferase (GLUC) is a bioluminescent reporter protein of increasing importance. As a secretory protein, it has increased sensitivity in vitro and in vivo (∼20 000-fold, and ∼1000-fold, respectively) over its competitor, secreted alkaline phosphatase. Unfortunately, this same advantageous secretory nature of GLUC limits its usefulness for many other possible intracellular applications, e.g., imaging signaling pathways in intact cells, in vivo imaging, and in developing molecular imaging biosensors to study protein-protein interactions and protein folding. Hence, to widen the research applications of GLUC, we developed engineered variants that increase its intracellular retention both by modifying the N-terminal secretory signal peptide and by tagging additional sequences to its C-terminal region. We found that when GLUC was expressed in mammalian cells, its N-terminal secretory signal peptide comprising amino acids 1-16 was essential for GLUC folding and functional activity in addition to its inherent secretory property. Modification of the C-terminus of GLUC by tagging a four amino acid (KDEL) endoplasmic reticulum targeting peptide in multiple repeats significantly improved its intracellular retention, with little impact on its folding and enzymatic activity. We used stable cells expressing this engineered GLUC with KDEL repeats to monitor chemically induced endoplasmic reticulum stress on cells. Additionally, we engineered an apoptotic sensor using modified variants of GLUC containing a four amino acid caspase substrate peptide (DEVD) between the GLUC protein and the KDEL repeats. Its use in cell culture resulted in increased GLUC secretion in the growth medium when cells were treated with the chemotherapeutic drugs doxorubicin, paclitaxel, and carboplatin. We thus successfully engineered a new variant GLUC protein that is retained inside cells rather than secreted extracellularly. We validated this novel reporter by incorporating it in biosensors for detection of cellular endoplasmic reticulum stress and caspase activation. This new molecularly engineered enzymatic reporter has the potential for widespread applications in biological research.

Fluorescent Lactic Acid Bacteria and Bifidobacteria as Vehicles of DNA Microbial Biosensors.

Control and quantification of effector molecules such as heavy metals, toxins or other target molecules is of great biotechnological, social and economic interest. Microorganisms have regulatory proteins that recognize and modify the gene expression in the presence or absence of these compounds (effector molecules) by means of binding to gene sequences. The association of these recognizing gene sequences to reporter genes will allow the detection of effector molecules of interest with high sensitivity. Once investigators have these two elements—recognizing gene sequences and reporter genes that emit signals—we need a suitable vehicle to introduce both elements. Here, we suggest lactic acid bacteria (LAB) and bifidobacteria as promising carrier microorganisms for these molecular biosensors. The use of fluorescent proteins as well as food-grade vectors and clustered regularly interspaced short palindromic repeats (CRISPR) are indispensable tools for introducing biosensors into these microorganisms. The use of these LAB and bifidobacteria would be of special interest for studying the intestinal environment or other complex ecosystems. The great variety of species adapted to many environments, as well as the possibility of applying several protocols for their transformation with recognizing gene sequences and reporter genes are considerable advantages. Finally, an effort must be made to find recognizable gene sequences.

Activatable interpolymer complex-superparamagnetic iron oxide nanoparticles as magnetic resonance contrast agents sensitive to oxidative stress

Magnetic resonance contrast agents that can be activated in response to specific triggers hold potential as molecular biosensors that may be of great utility in non-invasive disease diagnosis. We developed an activatable agent based on superparamagnetic iron oxide nanoparticles (SPIOs) that is sensitive to oxidative stress, a factor in the pathophysiology of numerous diseases. SPIOs were coated with poly(ethylene glycol) (PEG) and complexed with poly(gallol), a synthetic tannin. Hydrogen bonding between PEG and poly(gallol) creates a complexed layer around the SPIO that decreases the interaction of solute water with the SPIO, attenuating its magnetic resonance relaxivity. The complexed interpolymer nanoparticle is in an OFF state (decreased T2 contrast), where the contrast agent has a low T2 relaxivity of 7±2mM−1s−1. In the presence of superoxides, the poly(gallol) is oxidized and the polymers decomplex, allowing solute water to again interact with the SPIO, representing an ON state (increased T2 contrast) with a T2 relaxivity of 70±10mM−1s−1. These contrast agents show promise as effective sensors for diseases characterized in part by oxidative stress such as atherosclerosis, diabetes, and cancer.

Sensing Mechanisms in the Redox-Regulated, [2Fe-2S] Cluster-Containing, Bacterial Transcriptional Factor SoxR.

Bacteria possess molecular biosensors that enable responses to a variety of stressful conditions, including oxidative stress, toxic compounds, and interactions with other organisms, through elaborately coordinated regulation of gene expression. In Escherichia coli and related bacteria, the transcription factor SoxR functions as a sensor of oxidative stress and nitric oxide (NO). SoxR protein contains a [2Fe-2S] cluster essential for its transcription-enhancing activity, which is regulated by redox changes in the [2Fe-2S] cluster. We have explored the mechanistic and structural basis of SoxR proteins function and determined how the chemistry at the [2Fe-2S] cluster causes the subsequent regulatory response. In this Account, I describe our recent achievements in three different areas using physicochemical techniques, primarily pulse radiolysis. First, redox-dependent conformational changes in SoxR-bound DNA were studied by site-specifically replacing selected bases with the fluorescent probes 2-aminopurine and pyrrolocytosine. X-ray analyses of the DNA-SoxR complex in the oxidized state revealed that the DNA structure is distorted in the center regions, resulting in local untwisting of base pairs. However, the inactive, reduced state had remained uncharacterized. We found that reduction of the [2Fe-2S] cluster in the SoxR-DNA complex weakens the fluorescence intensity within a region confined to the central base pairs in the promoter region. Second, the reactions of NO with [2Fe-2S] clusters of E. coli SoxR were analyzed using pulse radiolysis. The transcriptional activation of SoxR in E. coli occurs through direct modification of [2Fe-2S] by NO to form a dinitrosyl iron complex (DNIC). The reaction of NO with [2Fe-2S] cluster of SoxR proceeded nearly quantitatively with concomitant reductive elimination of two equivalents S0 atoms. Intermediate nitrosylation products, however, were too unstable to observe. We found that the conversion proceeds through at least two steps, with the faster phase being the first reaction of the NO molecule with the [2Fe-2S] cluster. The slower reaction with the second equivalent NO molecule, however, was important for the formation of DNIC. Third, to elucidate the differences between the distinct responses of SoxR proteins from two different species, we studied the interaction of E. coli and Pseudomonas aeruginosa SoxR with superoxide anion using a mutagenic approach. Despite the homology between E. coli SoxR and P. aeruginosa SoxR, the function of P. aeruginosa SoxR differs from that of E. coli. The substitution of E. coli SoxR lysine residues, located close to [2Fe-2S] clusters, into P. aeruginosa SoxR dramatically affected the reaction with superoxide anion.

Design of a Toolbox of RNA Thermometers.

Biomolecular temperature sensors can be used for efficient control of large-volume bioreactors, for spatiotemporal imaging and control of gene expression, and to engineer robustness to temperature in biomolecular circuit design. Although RNA-based sensors, called "thermometers", have been investigated in both natural and synthetic contexts, an important challenge is to design diverse responses to temperature differing in sensitivity and threshold. We address this issue by constructing a library of RNA thermometers based on thermodynamic computations and experimentally measuring their activities in cell-free biomolecular "breadboards". Using free energies of the minimum free energy structures as well as melt profile computations, we estimated that a diverse set of temperature responses were possible. We experimentally found a wide range of responses to temperature in the range 29-37 °C with fold-changes varying over 3-fold around the starting thermometer. The sensitivities of these responses ranged over 10-fold around the starting thermometer. We correlated these measurements with computational expectations, finding that although there was no strong correlation for the individual thermometers, overall trends of diversity, fold-changes, and sensitivities were similar. These results present a toolbox of RNA-based circuit elements with diverse temperature responses.

Design and application of a lactulose biosensor

In this study the repressor of Escherichia coli lac operon, LacI, has been engineered for altered effector specificity. A LacI saturation mutagenesis library was subjected to Fluorescence Activated Cell Sorting (FACS) dual screening. Mutant LacI-L5 was selected and it is specifically induced by lactulose but not by other disaccharides tested (lactose, epilactose, maltose, sucrose, cellobiose and melibiose). LacI-L5 has been successfully used to construct a whole-cell lactulose biosensor which was then applied in directed evolution of cellobiose 2-epimerase (C2E) for elevated lactulose production. The mutant C2E enzyme with ~32-fold enhanced expression level was selected, demonstrating the high efficiency of the lactulose biosensor. LacI-L5 can also be used as a novel regulatory tool. This work explores the potential of engineering LacI for customized molecular biosensors which can be applied in practice.

Multiple Schottky Barrier-Limited Field-Effect Transistors on a Single Silicon Nanowire with an Intrinsic Doping Gradient.

In comparison to conventional (channel-limited) field-effect transistors (FETs), Schottky barrier-limited FETs possess some unique characteristics which make them attractive candidates for some electronic and sensing applications. Consequently, modulation of the nano Schottky barrier at a metal-semiconductor interface promises higher performance for chemical and biomolecular sensor applications when compared to conventional FETs with ohmic contacts. However, the fabrication and optimization of devices with a combination of ideal ohmic and Schottky contacts as the source and drain, respectively, present many challenges. We address this issue by utilizing Si nanowires (NWs) synthesized by a chemical vapor deposition process which yields a pronounced doping gradient along the length of the NWs. Devices with a series of metal contacts on a single Si NW are fabricated in a single lithography and metallization process. The graded doping profile of the NW is manifested in monotonic increases in the channel and junction resistances and variation of the nature of the contacts from ohmic to Schottky of increasing effective barrier height along the NW. Hence multiple single Schottky junction-limited FETs with extreme asymmetry and high reproducibility are obtained on an individual NW. A definitive correlation between increasing Schottky barrier height and enhanced gate modulation is revealed. Having access to systematically varying Schottky barrier contacts on the same NW device provides an ideal platform for identifying optimal device characteristics for sensing and electronic applications.

Fabrication of Nanopillar-Based Split Ring Resonators for Displacement Current Mediated Resonances in Terahertz Metamaterials

Terahertz (THz) split ring resonator (SRR) metamaterials (MMs) has been studied for gas, chemical, and biomolecular sensing applications because the SRR is not affected by environmental characteristics such as the temperature and pressure surrounding the resonator. Electromagnetic radiation in THz frequencies is biocompatible, which is a critical condition especially for the application of the biomolecular sensing. However, the quality factor (Q-factor) and frequency responses of traditional thin-film based split ring resonator (SRR) MMs are very low, which limits their sensitivities and selectivity as sensors. In this work, novel nanopillar-based SRR MMs, utilizing displacement current, are designed to enhance the Q-factor up to 450, which is around 45 times higher than that of traditional thin-film-based MMs. In addition to the enhanced Q-factor, the nanopillar-based MMs induce a larger frequency shifts (17 times compared to the shift obtained by the traditional thin-film based MMs). Because of the significantly enhanced Q-factors and frequency shifts as well as the property of biocompatible radiation, the THz nanopillar-based SRR are ideal MMs for the development of biomolecular sensors with high sensitivity and selectivity without inducing damage or distortion to biomaterials. A novel fabrication process has been demonstrated to build the nanopillar-based SRRs for displacement current mediated THz MMs. A two-step gold (Au) electroplating process and an atomic layer deposition (ALD) process are used to create sub-10 nm scale gaps between Au nanopillars. Since the ALD process is a conformal coating process, a uniform aluminum oxide (Al2O3) layer with nanometer-scale thickness can be achieved. By sequentially electroplating another Au thin film to fill the spaces between Al2O3 and Au, a close-packed Au-Al2O3-Au structure with nano-scale Al2O3 gaps can be fabricated. The size of the nano-gaps can be well defined by precisely controlling the deposition cycles of the ALD process, which has an accuracy of 0.1 nm.

Fabrication of Nanopillar-Based Split Ring Resonators for Displacement Current Mediated Resonances in Terahertz Metamaterials

Terahertz (THz) split ring resonator (SRR) metamaterials (MMs) has been studied for gas, chemical, and biomolecular sensing applications because the SRR is not affected by environmental characteristics such as the temperature and pressure surrounding the resonator. Electromagnetic radiation in THz frequencies is biocompatible, which is a critical condition especially for the application of the biomolecular sensing. However, the quality factor (Q-factor) and frequency responses of traditional thin-film based split ring resonator (SRR) MMs are very low, which limits their sensitivities and selectivity as sensors. In this work, novel nanopillar-based SRR MMs, utilizing displacement current, are designed to enhance the Q-factor up to 450, which is around 45 times higher than that of traditional thin-film-based MMs. In addition to the enhanced Q-factor, the nanopillar-based MMs induce a larger frequency shifts (17 times compared to the shift obtained by the traditional thin-film based MMs). Because of the significantly enhanced Q-factors and frequency shifts as well as the property of biocompatible radiation, the THz nanopillar-based SRR are ideal MMs for the development of biomolecular sensors with high sensitivity and selectivity without inducing damage or distortion to biomaterials. A novel fabrication process has been demonstrated to build the nanopillar-based SRRs for displacement current mediated THz MMs. A two-step gold (Au) electroplating process and an atomic layer deposition (ALD) process are used to create sub-10 nm scale gaps between Au nanopillars. Since the ALD process is a conformal coating process, a uniform aluminum oxide (Al2O3) layer with nanometer-scale thickness can be achieved. By sequentially electroplating another Au thin film to fill the spaces between Al2O3 and Au, a close-packed Au-Al2O3-Au structure with nano-scale Al2O3 gaps can be fabricated. The size of the nano-gaps can be well defined by precisely controlling the deposition cycles of the ALD process, which has an accuracy of 0.1 nm.