Detection Of Single Biomolecules Research Articles

A Quantitative Description for Optical Mass Measurement of Single Biomolecules.

Label-free detection of single biomolecules in solution has been achieved using a variety of experimental approaches over the past decade. Yet, our understanding of the magnitude of the optical contrast and its relationship with the underlying atomic structure as well as the achievable measurement sensitivity and precision remain poorly defined. Here, we use a Fourier optics approach combined with an atomic structure-based molecular polarizability model to simulate mass photometry experiments from first principles. We find excellent agreement between several key experimentally determined parameters such as optical contrast-to-mass conversion, achievable mass accuracy, and molecular shape and orientation dependence. This allows us to determine detection sensitivity and measurement precision mostly independent of the optical detection approach chosen, resulting in a general framework for light-based single-molecule detection and quantification.

Single‐Molecule Electronic Biosensors: Principles and Applications (Adv. Sensor Res. 5/2023)

Single-Molecule Electronic Biosensors In article number 2200084, Jinying Wang, Chuancheng Jia, Xuefeng Guo, and co-workers summarize the working principles, device architectures, and potential applications of single-molecule electronic biosensors for near-field and real-time detection of single biomolecules, and provide a promising optoelectronic integrated system for realizing synchronous acquisition of electrical and optical signals of single biomolecules.

Nanotechnology-Enabled Biosensors: A Review of Fundamentals, Design Principles, Materials, and Applications.

Biosensors are modern engineering tools that can be widely used for various technological applications. In the recent past, biosensors have been widely used in a broad application spectrum including industrial process control, the military, environmental monitoring, health care, microbiology, and food quality control. Biosensors are also used specifically for monitoring environmental pollution, detecting toxic elements' presence, the presence of bio-hazardous viruses or bacteria in organic matter, and biomolecule detection in clinical diagnostics. Moreover, deep medical applications such as well-being monitoring, chronic disease treatment, and in vitro medical examination studies such as the screening of infectious diseases for early detection. The scope for expanding the use of biosensors is very high owing to their inherent advantages such as ease of use, scalability, and simple manufacturing process. Biosensor technology is more prevalent as a large-scale, low cost, and enhanced technology in the modern medical field. Integration of nanotechnology with biosensors has shown the development path for the novel sensing mechanisms and biosensors as they enhance the performance and sensing ability of the currently used biosensors. Nanoscale dimensional integration promotes the formulation of biosensors with simple and rapid detection of molecules along with the detection of single biomolecules where they can also be evaluated and analyzed critically. Nanomaterials are used for the manufacturing of nano-biosensors and the nanomaterials commonly used include nanoparticles, nanowires, carbon nanotubes (CNTs), nanorods, and quantum dots (QDs). Nanomaterials possess various advantages such as color tunability, high detection sensitivity, a large surface area, high carrier capacity, high stability, and high thermal and electrical conductivity. The current review focuses on nanotechnology-enabled biosensors, their fundamentals, and architectural design. The review also expands the view on the materials used for fabricating biosensors and the probable applications of nanotechnology-enabled biosensors.

Photoswitchable Binary Nanopore Conductance and Selective Electronic Detection of Single Biomolecules under Wavelength and Voltage Polarity Control.

We fabricated photoregulated thin-film nanopores by covalently linking azobenzene photoswitches to silicon nitride pores with ∼10 nm diameters. The photoresponsive coatings could be repeatedly optically switched with deterministic ∼6 nm changes to the effective nanopore diameter and of ∼3× to the nanopore ionic conductance. The sensitivity to anionic DNA and a neutral complex carbohydrate biopolymer (maltodextrin) could be photoswitched "on" and "off" with an analyte selectivity set by applied voltage polarity. Photocontrol of nanopore state and mass transport characteristics is important for their use as ionic circuit elements (e.g., resistors and binary bits) and as chemically tuned filters. It expands single-molecule sensing capabilities in personalized medicine, genomics, glycomics, and, augmented by voltage polarity selectivity, especially in multiplexed biopolymer information storage schemes. We demonstrate repeatedly photocontrolled stable nanopore size, polarity, conductance, and sensing selectivity, by illumination wavelength and voltage polarity, with broad utility including single-molecule sensing of biologically and technologically important polymers.

Multi-layered heterostructure nanopore sensor for single biomolecule detection

Nanopores have shown to be a promising tool for fast and real-time sequencing of DNA. Due to the chemical and mechanical robustness and design flexibility, two-dimensional (2D) solid-state nanopore (SSNP) sensors are a contesting candidate for next-generation sequencing devices. However, the major hindrance in detecting DNA nucleotides via 2D SSNPs lies in the low signal-to-noise ratio (SNR) due to thermal noise and the lack of statistically significant current signals. In this regard, we propose a stacked 2D SSNP heterostructure to naturally quench the conformational fluctuations of the translocating biomolecule while obtaining multiple independent electronic in-plane current signals.

Digital Counting of Biomolecules Using Engineered Functional DNA Superstructures.

There is currently a great need for developing a simple and effective biosensing platform for the detection of single biomolecules (e.g., DNAs, RNAs, or proteins) in the biological, medical, and environmental fields. Here, we show a versatile and sensitive fluorescence counting strategy for quantifying proteins and microRNAs by employing functional DNA superstructures (denoted as 3D DNA). A 3D DNA biolabel was first engineered to become highly fluorescent and carry recognition elements for the target of interest. The presence of a target cross-links the resultant of the 3D DNA biolabel and a surface-bound capturing antibody or DNA oligonucleotide, thus forming a sandwich complex that can be easily resolved using traditional fluorescence microscopy. The broad utility of this platform is illustrated by engineering two different 3D DNA biolabels that enable the quantification of β-lactamase (one secreted bacterial hydrolase) and miR-21 (one overexpressed microRNA in cancer cells) with detection limits of 100 aM and 1 fM, respectively. We envision that the approach described herein will find useful applications in chemical biology, medical diagnostics, and biosensing.

Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy.

Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.

Ultrasensitive Electrochemiluminescence Biosensor Based on Closed Bipolar Electrode for Alkaline Phosphatase Detection in Single Liver Cancer Cell.

An ultrasensitive electrochemiluminescence (ECL) biosensor was proposed based on a closed bipolar electrode (BPE) for the detection of alkaline phosphatase (ALP). For most of the BPE-ECL biosensors, an effective signal amplification strategy was the key to enhance the sensitivity of the system. Herein, the signal amplification strategy of the enzyme catalysis was utilized in the BPE-ECL system. Au nanoparticles (NPs) were electrodeposited on the cathode surface of the ITO electrode to improve the stability and sensitivity of the signal. Compared with the previous BPE-ECL biosensors, the sensitivity was increased by at least 3 orders of magnitude. The biosensor showed high sensitivity and specificity of ALP detection with a detection limit of as low as 3.7 aM. Besides, it was further applied to the detection of ALP in different types of cells and successfully realized ALP detection in single Hep G2 cell, which had a huge application prospect in single biomolecule detection or single cell analysis.

Microscopic Detection Analysis of Single Molecules in MoS2 Membrane Nanopores.

A systematic microscopic analysis of the various resistive effects involved in the electronic detection of single biomolecules in a nanopore of a MoS2 nanoribbon is presented. The variations of the transverse electronic current along the two-dimensional (2D) membrane due to the translocation of DNA and protein molecules through the pore are obtained by model calculations based on molecular dynamics (MD) and Boltzmann transport formalism, which achieved good agreement with the experimental data. Our analysis points to a self-consistent interaction among ions, charge carriers around the pore rim, and biomolecules. It provides a comprehensive understanding of the effects of the electrolyte concentration, pore size, nanoribbon geometry, and also the doping polarity of the nanoribbon on the electrical sensitivity of the nanopore in detecting biomolecules. These results can be utilized for fine-tuning the design parameters in the fabrication of highly sensitive 2D nanopore biosensors.

Nanogap-based all-electronic DNA sequencing devices using MoS2 monolayers.

The realization of nanopores in atom-thick materials may pave the way towards electrical detection of single biomolecules in a stable and scalable manner. In this work, we theoretically study the potential of different phases of MoS2 nanogaps to act as all-electronic DNA sequencing devices. We carry out simulations based on density functional theory and the non-equilibrium Green's function formalism to investigate the electronic transport across the device. Our results suggest that the 1T'-MoS2 nanogap structure is energetically more favorable than its 2H counterpart. At zero bias, the changes in the conductance of the 1T'-MoS2 device can be well distinguished, making possible the selectivity of the DNA nucleobases. Although the conductance fluctuates around the resonances, the overall results suggest that it is possible to distinguish the four DNA bases for energies close to the Fermi level.

Plasmon-enhanced stimulated Raman scattering microscopy with single-molecule detection sensitivity

Stimulated Raman scattering (SRS) microscopy allows for high-speed label-free chemical imaging of biomedical systems. The imaging sensitivity of SRS microscopy is limited to ~10 mM for endogenous biomolecules. Electronic pre-resonant SRS allows detection of sub-micromolar chromophores. However, label-free SRS detection of single biomolecules having extremely small Raman cross-sections (~10−30 cm2 sr−1) remains unreachable. Here, we demonstrate plasmon-enhanced stimulated Raman scattering (PESRS) microscopy with single-molecule detection sensitivity. Incorporating pico-Joule laser excitation, background subtraction, and a denoising algorithm, we obtain robust single-pixel SRS spectra exhibiting single-molecule events, verified by using two isotopologues of adenine and further confirmed by digital blinking and bleaching in the temporal domain. To demonstrate the capability of PESRS for biological applications, we utilize PESRS to map adenine released from bacteria due to starvation stress. PESRS microscopy holds the promise for ultrasensitive detection and rapid mapping of molecular events in chemical and biomedical systems.

Intelligent Quantification of Picomolar Protein Concentration in Serum by Functionalized Nanopores

Nanopores have been well established as a promising platform for real time stochastic detection of single biomolecules and have made sufficient commercial progress in terms of DNA sequencing. Amongst the various strategies for specific protein estimation in physiological analyte, aptamer functionalized nanopores have been reported to quantify proteins down to few picomolars in control solution. In this paper, we explore the quantification of target protein in serum down to picomolar concentration using aptamer functionalized nanopores. For such cases, the current settles to a new value in multiple steps due to the low dissociation constants of the receptors and the final current blockade sensitivity is the primary indicator of target protein concentration. It has been observed that the current sensitivity histograms not only have a statistical variation (due to the fluctuations in the device fabrication) but also overlap significantly between the different concentration ranges in the picomolar regime, which makes quantification challenging. Here, we introduce probabilistic fuzzy model based on Monte Carlo simulation and demonstrate its ability by quantifying thrombin down to 50-pM concentration in undiluted serum. The method has been verified with 25 test solutions and the results reveal the potential of this computational approach toward lowering the detection limit by three orders of magnitude compared with the existing status, thus enabling the functionalized glass nanopore platform makes great progress toward clinical testing.

Guest Editorial Special Issue on Advances and Open Challenges for Integrated Circuits Detecting Bio Molecules

The four articles in this special section focus on multidisciplinary advancements for CMOS integrated circuit technology-based solutions. Specially, examines advances for integrated circuits that detect biomolecules. Despite the continuing convergence of microelectronics with biomedicine, important technical challenges still constrain the deployment of integrated circuits in microanalytical devices, operating both in vivo and in vitro. From the standpoint of electronic circuit design, the trade-offs between resolution (noise), area (cost), and power dissipation need to be carefully optimized. These tradeoffs, while familiar, are crucial for endowing portable, wearable, and implantable devices with high sensitivity. Some applications prioritize ultra low noise, such as single-biomolecule detection using nanopore sensors. In other spaces, power dissipation and die area are clearly major constraints when thousands of sensing channels are integrated on a single chip, as in the case of image sensors and electrophysiology arrays interfacing with live tissue. Secondly, from a system-level point of view, reliable and scalable solutions are required for packaging CMOS chips to be used in contact with liquid samples. Fabrication approaches need to be found for creating chip-to-liquid interfaces (micro-tomacro scale), which are feasible both for small-scale academic prototypes and for high-volume manufacturing. Other perennial challenges include post-silicon fabrication of high performance transducers and electrodes. Finally, the different avenues for monolithic or hybrid integration of multichannel and low-noise analog front-ends with digital back-ends for data processing and wireless communication circuitry pose important technical design challenges.

There and back again: from the origin of life to single molecules

What is life? There is hardly a more fundamental question raised by aspiring researchers, and one less prone to ever be answered in a scientifically satisfying way. In the long, productive and highly influential period of research following his Nobel-recognised work on relaxation kinetics, Manfred Eigen made seminal contributions towards a quantifiable definition of life, with a strong focus on its evolutionary character. In the last years of his time as an active researcher, however, he devoted himself to another, purely experimental topic: the detection and analysis of single biomolecules in aqueous solution. In this short review, I will give an overview of the groundbreaking contributions to the field of single molecule research made by Eigen and coworkers, and show that both, in its intrinsic motivation, and in its consequences, single molecule research strongly relates to the question of the physical–chemical essence of life. In fact, research on living systems with single molecule sensitivity will always refer the researcher to the question of the simplest possible representation, and thus the origin, of any biological phenomenon.

Applications of a hybrid photodetector (HPD) to biological fluorescence microscopes and wide-field fluorescence detection of mobile single biomolecules with high temporal resolution using an HPD

Applications of a hybrid photodetector (HPD) to biological fluorescence microscopes and wide-field fluorescence detection of mobile single biomolecules with high temporal resolution using an HPD

Single-Molecule Characterization of DNA-Protein Interactions Using Nanopore Biosensors.

Detection and characterization of nucleic acid-protein interactions, particularly those involving DNA and proteins such as transcription factors, enzymes, and DNA packaging proteins, remain significant barriers to our understanding of genetic regulation. Nanopores are an extremely sensitive and versatile sensing platform for label-free detection of single biomolecules. Analyte molecules are drawn to and through a nanoscale aperture by an electrophoretic force, which acts upon their native charge while in the sensing region of the pore. When the nanopore's diameter is only slightly larger than the biopolymer's cross section (typically a few nm); the latter must translocate through the pore in a linear fashion due to the constricted geometry in this region. These features allow nanopores to interrogate protein-nucleic acids in multiple sensing modes: first, by scanning and mapping the locations of binding sites along an analyte molecule, and second, by probing the strength of the bond between a protein and nucleic acid, using the native charge of the nucleic acid to apply an electrophoretic force to the complex while the protein is geometrically prevented from passing through the nanopore. In this chapter, we describe progress toward nanopore sensing of protein-nucleic acid complexes in the context of both mapping binding sites and performing force spectroscopy to determine the strength of interactions. We conclude by reviewing the strengths and challenges of the nanopore technique in the context of studying DNA-protein interactions.

Manipulation and detection of single nanoparticles and biomolecules by a photonic nanojet.

Optical methods to manipulate and detect nanoscale objects are highly desired in both nanomaterials and molecular biology fields. Optical tweezers have been used to manipulate objects that range in size from a few hundred nanometres to several micrometres. The emergence of near-field methods that overcome the diffraction limit has enabled the manipulation of objects below 100 nm. A highly free manipulation with signal-enhanced real-time detection, however, remains a challenge for single sub-100-nm nanoparticles or biomolecules. Here we show an approach that uses a photonic nanojet to perform the manipulation and detection of single sub-100-nm objects. With the photonic nanojet generated by a dielectric microlens bound to an optical fibre probe, three-dimensional manipulations were achieved for a single 85-nm fluorescent polystyrene nanoparticle as well as for a plasmid DNA molecule. Backscattering and fluorescent signals were detected with the enhancement factors up to ∼103 and ∼30, respectively. The demonstrated approach provides a potentially powerful tool for nanostructure assembly, biosensing and single-biomolecule studies.

Recent Advances in Point of Care Diagnostic Tools: A Review

Globally, there is a great need for portable, inexpensive diagnostic tools that can provide quick, accurate results using relatively small sample volumes. Point-Of-Care (POC) measurements of human saliva, sweat and/or blood capable of detecting glucose levels, foreign pathogens such as bacteria, fungi and many different viruses (HIV, Ebola, Influenza, etc.) that use microfluidics and optofluidics are close to commercial availability. These diagnostic tools use both optical and electrical methods for the detection and analysis of single biomolecules. The applications extend beyond healthcare and can be used for pathogen detection in aquatic environments such as drinking water. Additional research in handheld Optical Coherence Tomography (OCT) offers the possibility for inexpensive diagnosis and treatment of tissue like middle ear infection and breast cancer. Finally, microscopes are also getting smaller and cheaper, with an inexpensive plastic florescence microscope that can quantify white blood cell levels and a suitcase sized microscope that can look at a single drop of blood for a million biomarkers, searching for signs of sepsis and infectious disease. POC devices such as these will provide medical care to poor, remote areas that can be administered by junior physicians or even used for self-diagnosis. In this study, a review of advances in POC devices reported in recent literature is conducted, in order to provide the reader with a thorough description of new diagnostic techniques that have taken place in the last couple of years.

Tuneable graphene nanopores for single biomolecule detection.

Solid-state nanopores are promising candidates for next generation DNA and protein sequencing. However, once fabricated, such devices lack tuneability, which greatly restricts their biosensing capabilities. Here we propose a new class of solid-state graphene-based nanopore devices that exhibit a unique capability of self-tuneability, which is used to control their conductance, tuning it to levels comparable to the changes caused by the translocation of a single biomolecule, and hence, enabling high detection sensitivities. Our presented quantum simulation results suggest that the smallest amino acid, glycine, when present in water and in an aqueous saline solution can be detected with high sensitivity, up to a 90% change in conductance. Our results also suggest that passivating the device with nitrogen, making it an n-type device, greatly enhances its sensitivity, and makes it highly sensitive to not only the translocation of a single biomolecule, but more interestingly to intramolecular electrostatics within the biomolecule. Sensitive detection of the carboxyl group within the glycine molecule, which carries a charge equivalent to a single electron, is achieved with a conductance change that reaches as high as 99% when present in an aqueous saline solution. The presented findings suggest that tuneable graphene nanopores, with their capability of probing intramolecular electrostatics, could pave the way towards a new generation of single biomolecule detection devices.

Optofluidic wavelength division multiplexing for single-virus detection

Optical waveguides simultaneously transport light at different colors, forming the basis of fiber-optic telecommunication networks that shuttle data in dozens of spectrally separated channels. Here, we reimagine this wavelength division multiplexing (WDM) paradigm in a novel context--the differentiated detection and identification of single influenza viruses on a chip. We use a single multimode interference (MMI) waveguide to create wavelength-dependent spot patterns across the entire visible spectrum and enable multiplexed single biomolecule detection on an optofluidic chip. Each target is identified by its time-dependent fluorescence signal without the need for spectral demultiplexing upon detection. We demonstrate detection of individual fluorescently labeled virus particles of three influenza A subtypes in two implementations: labeling of each virus using three different colors and two-color combinatorial labeling. By extending combinatorial multiplexing to three or more colors, MMI-based WDM provides the multiplexing power required for differentiated clinical tests and the growing field of personalized medicine.