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Tunable nanochannel resistive pulse sensing device using a novel multi-module self-assembly
Abstract
Nanochannel-based resistive pulse sensing (nano-RPS) system is widely used for the high-sensitive measurement and characterization of nanoscale biological particles and biomolecules due to its high surface to volume ratio. However, the geometric dimensions and surface properties of nanochannel are usually fixed, which limit the detections within particular ranges or types of nanoparticles. In order to improve the flexibility of nano-RPS system, it is of great significance to develop nanochannels with tunable dimensions and surface properties. In this work, we proposed a novel multi-module self-assembly (MS) strategy which allows to shrink the geometric dimensions and tune surface properties of the nanochannels simultaneously. The MS-tuned nano-RPS device exhibits an enhanced signal-to-noise ratio (SNR) for nanoparticle detections after shrunk the geometric dimensions by MS strategy. Meanwhile, by tuning the surface charge, an enhanced resolution for viral particles detection was achieved with the MS-tuned nano-RPS devices by analyzing the variation of pulse width due the tuned surface charge. The proposed MS strategy is versatile for various types of surface materials and can be potentially applied for nanoscale surface reconfiguration in various nanofluidic devices.
... The interaction between analytes and the self-assembled layer would change the surface chemical proper, changing the ion current through the channel. This method has been used for detecting chiral amino acids, proteins, and viruses [39][40][41]. ...
Nanopipette-based sensors are one of the most effective tools for detecting nanoparticles, bioparticles, and biomolecules. Quantitative analysis of nanoparticles with different shapes and electrical charges is achieved through measurement of the blockage currents that occur when particles pass through the nanopore. However, typical nanopipette sensors fabricated using a conventional needle-pulling method have a typical pore-diameter limitation of around 100 nm. Herein, we report a novel conductive hydrogel-composited nanopipette sensor with a tunable inner-pore diameter. This is made by electrodepositing poly(3,4-ethylenedioxythiophene) polystyrene sulfonate onto the surface of a nanopipette with a prefabricated sacrificial copper layer. Because of the presence of copper ions, the conductive polymer can stably adhere to the tip of the nanopipette to form a nanopore; when nanoparticles pass through the conductive nanopore, more distinct blocking events are observed. The size of the nanopore can be changed simply by adjusting the electrodeposition time. In this way, suitable nanopores can be obtained for highly sensitive screening of a series of particles with diameters of the order of tens of nanometers.
Microfluidic resistive pulse sensing (MRPS) was used to determine the size distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) based on detecting nearly 30 000 single virions. However, the ultrastructure of SARS-CoV-2 is thoroughly described, but ensemble properties of SARS-CoV-2, e.g., its particle size distribution, are sparsely reported. According to the MRPS results, the size distribution of SARS-CoV-2 follows a log-normal function with a mean value of 85.1 nm, which corresponds to an approximate diameter of the viral envelope. This result also confirms the low number (< 50) of spike proteins on the surface of the virions.
Several vaccines against COVID-19 use nanoparticles to protect the antigen cargo (either proteins or nucleic acids), increase the immunogenicity and ultimately the efficacy. The characterization of these nanomedicines is challenging due to their intrinsic complexity and requires the use of multidisciplinary techniques and competencies. The accurate characterization of nanovaccines can be conceptualized as a combination of physicochemical, immunological and toxicological assays. This will help to address key challenges in the preclinical characterization, will guide the rapid development of safe and effective vaccines for current and future health crises, and will streamline the regulatory process. This Perspective suggests a characterization cascade for nanoparticle-based vaccines that takes into consideration the complexity of the materials and of their immunomodulatory responses, providing a roadmap for the preclinical validation of potential vaccine candidates.
Inspired by the design and performance of biological pores, polymer functionalization of nanopores has emerged as an evolving field to advance transport performance within the last few years. This feature article outlines developments in nanopore functionalization and the resulting transport performance including gating based on electrostatic interaction, wettability and ligand binding, gradual transport controlled by polymerization as well as functionalization-based asymmetric nanopore and nanoporous material design going towards the transport direction. Pushing the limits of nanopore transport performance and thus reducing the performance gap between biological and technological pores is strongly related to advances in polymerization chemistry and their translation into nanopore functionalization. Thereby, the effect of the spatial confinement has to be considered for polymer functionalization as well as for transport regulation, and mechanistic understanding is strongly increased by combining experiment and theory. A full mechanistic understanding together with highly precise nanopore structure design and polymer functionalization is not only expected to improve existing application of nanoporous materials but also opens the door to new technologies. The latter might include out of equilibrium devices, ionic circuits, or machine learning based sensors.
In situ labelling and encapsulation of biological entities, such as of single viruses, may provide a versatile approach to modulate their functionality and facilitate their detection at single particle level. Here, we introduce a novel virus metallization approach based on in situ coating of viruses in solution with silver nanoparticles (AgNP) in a two-step synthetic process, i.e. surface activation with a tannic acid - Sn(II) coordination complex, which subsequently induces silver ion (I) reduction. The metalic coating on the virus surface opens the opportunity for electrochemical quantification of the AgNP-tagged viruses by nano-impact electrochemistry on a microelectrode with single particle sensitivity, i.e. enable the detection of particles oherwise undetectable. We show that the silver coating of the virus particles impacting the electrode can be oxidized to produce distinct current peaks the frequency of which show a linear correlation with the virus count. The proof of the concept was done with inactivated Influenza A (H3N2) viruses resulting in their quantitation down to the femtomolar concentrations (ca. 5 × 107 particles per mL) using 50 s counting sequences.
he ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has killed untold millions worldwide and has hurtled vaccines into the spotlight as a go-to approach to mitigate it. Advances in virology, genomics, structural biology, and vaccine technologies enabled a rapid and unprecedented rollout of COVID-19 vaccines, although much of the developing world remains unvaccinated. Several new vaccine platforms have been developed or deployed against SARS-CoV-2, with most targeting the large viral Spike immunogen. Those that safely induce strong and durable antibody responses at low dosages are advantageous, as well are those that can be rapidly produced at a large scale. Virtually all COVID-19 vaccines and adjuvants possess nanoscale or microscale dimensions and represent diverse and unique biomaterials. Viral vector vaccine platforms, lipid nanoparticle mRNA vaccines and multimeric display technologies for subunit vaccines have received much attention. Nanoscale vaccine adjuvants have also been used in combination with other vaccines. To deal with the ongoing pandemic, and to be ready for potential future ones, advanced vaccine technologies will continue to be developed in the near future. In this review, we summarize recent use of advanced materials used for developing COVID-19 vaccines.
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This paper reports an IC-compatible method for fabricating a PDMS-based resistive pulse sensing (RPS) device with embedded nanochannel (nanochannel-RPS) for label-free analysis of biomolecules and bionanoparticles, such as plasmid DNAs and exosomes. Here, a multilayer lithography process was proposed to fabricate the PDMS mold for the microfluidic device comprising a bridging nanochannel, as the sensing gate. RPS was performance by placing the sensing and excitation electrodes symmetrically upstream and downstream the sensing gate. In order to reduce the noise level, a reference electrode was designed and placed beside the excitation electrode. To demonstrate the feasibility of the proposed nanochannel-RPS device and sensing system, polystyrene micro- and nanoparticles with diameters of 1 μm and 300 nm were tested by the proposed device with signal-to-noise ratios (SNR) ranging 9.1-30.5 and 2.2-5.9, respectively. Furthermore, nanochannel with the height of 300 nm was applied for 4 kb plasmid DNA detection, implying the potential of the proposed method for label-free quantification of nanoscale biomolecules. Moreover, HeLa cell exosomes, known as a well-studied subtype of extracellular vesicles, were measured and analyzed by their size distribution. The result of the resistive pulse amplitude was well corresponding to the that of nanoparticle tracking analysis (NTA). The proposed nanochannel-RPS device and the sensing strategy are not only capable for label-free analysis for nanoscale biomolecules and bionanoparticle, but also cost-effective for large-scale manufacturing.
Nanofluidics, a discipline of science and engineering of fluids confined to structures at the 1-1000 nm scale, has experienced significant growth over the past decade. Nanofluidics have offered fascinating platforms for chemical and biological analyses by exploiting the unique characteristics of liquids and molecules confined in nanospaces; however, the difficulty to detect molecules in extremely small spaces hampers the practical applications of nanofluidic devices. Laser-induced fluorescence microscopy with single-molecule sensitivity has been so far a major detection method in nanofluidics, but issues arising from labeling and photobleaching limit its application. Recently, numerous label-free detection methods have been developed to identify and determine the number of molecules, as well as provide chemical, conformational, and kinetic information of molecules. This review focuses on label-free detection techniques designed for nanofluidics; these techniques are divided into two groups: optical and electrical/electrochemical detection methods. In this review, we discuss on the developed nanofluidic device architectures, elucidate the mechanisms by which the utilization of nanofluidics in manipulating molecules and controlling light-matter interactions enhances the capabilities of biological and chemical analyses, and highlight new research directions in the field of detections in nanofluidics.
Laser spectroscopy outperforms electrochemical and semiconductor gas sensors in selectivity and environmental survivability. However, the performance of the state-of-the-art laser sensors is still insufficient for many high precision applications. Here, we report mode-phase-difference photothermal spectroscopy with a dual-mode anti-resonant hollow-core optical fiber and demonstrate all-fiber gas (acetylene) detection down to ppt (parts-per-trillion) and <1% instability over a period of 3 hours. An anti-resonant hollow-core fiber could be designed to transmit light signals over a broad wavelength range from visible to infrared, covering molecular absorption lines of many important gases. This would enable multi-component gas detection with a single sensing element and pave the way for ultra-precision gas sensing for medical, environmental and industrial applications.
Although resistive pulse sensing using solid state nanopores is capable of single molecule sensitivity, previous work has shown that measurements can often significantly deviate from theoretically predicted current amplitudes and event frequencies, because most nanoparticles pass through the pore too quickly for accurate detection or resolution by typical measurement apparatus. Here, we show that a hydrogel placed on the distal side of a nanopore can increase the residence time of nanoparticles within the nanopore, significantly increasing the detection rate and allowing improved resolution of blockage currents. The method is simple and inexpensive to implement while being label-free and applicable to a wide range of nanoparticle targets. Using hydrogel-backed nanopores, we detected the protein IgG with event frequencies several orders of magnitude higher than in the absence of the hydrogel, and with larger measured currents that agree well with theoretical models. We also show that the improved measurement also enables discrimination of IgG and BSA in a mixed solution. Lastly, we show that measurements of IgG with the hydrogel-backed nanopores can also yield current amplitude distributions that can be analyzed to infer its approximate shape.
The signature events caused by host-guest interactions in the nanopore system can be used as a novel and characteristic signal in quantitative detection and analysis of various molecules. However, the effect of several electrochemical factors on the host-guest interactions in nanopore still remains unknown. Here, we systematically studied host-guest interactions, especially oscillation of DNA-azide [email protected][6] in α-Hemolysin nanopore under varying pH, concentration of electrolytes and counterions (Li⁺, Na⁺, K⁺). Our results indicate correlations between the change of pH and the duration of the oscillation signal. In addition, the asymmetric electrolyte concentration and the charge of the counterions affects the frequency of signature events in oscillation signals, and even the integrity of the protein nanopore. This study provides insight into the design of a future biosensing platform based on signature oscillation signals of the host-guest interaction within a nanopore.
Solid-state nanopores are single-molecule sensors that hold great potential for rapid protein and nucleic-acid analysis. Despite their many opportunities, the conventional ionic current detection scheme that is at the heart of the sensor suffers inherent limitations. This scheme intrinsically couples signal strength to the driving voltage, requires the use of high-concentration electrolytes, suffers from capacitive noise, and impairs high-density sensor integration. Here, we propose a fundamentally different detection scheme based on the enhanced light transmission through a plasmonic nanopore. We demonstrate that translocations of single DNA molecules can be optically detected, without the need of any labeling, in the transmitted light intensity through an inverted-bowtie plasmonic nanopore. Characterization and the cross-correlation of the optical signals with their electrical counterparts verify the plasmonic basis of the optical signal. We demonstrate DNA translocation event detection in a regime of driving voltages and buffer conditions where traditional ionic current sensing fails. This label-free optical detection scheme offers opportunities to probe native DNA-protein interactions at physiological conditions.
Cancer biomarkers are expected to be indicative of the occurrence of certain cancer diseases before the tumors form and metastasize. However, many biomarkers can only be acquired in extremely low concentrations, which are often beyond the limit of detection (LOD) of current instruments and technologies. A practical strategy for nanopore sensing of cancer biomarkers in raw human blood down to the femtomolar level is developed here. This strategy first converts the detection of cancer biomarkers to the quantification of copper ions by conducting a sandwich assay involving copper oxide nanoparticles. The released Cu²⁺ is then taken to catalyze the “click” reaction which ligates a host–guest modified DNA probe. Finally, this DNA probe is subjected to single‐channel recordings to afford the translocation events that can be used to derive the concentrations of the original biomarkers. Due to the amplification effects of nanoparticle loadings and the “click” reaction, the LOD of this strategy can be as low as the subfemtomolar level. Further, the acid treatment step could effectively eliminate the interferences from plasma proteins in raw human blood and make the strategy highly suitable for the detection of cancer biomarkers in clinical samples.
Starting from the background of nanofluidics in other disciplines, this paper describes the present state of research in this field and discusses possible directions of development. Emphasis is put on the very diverse background of nanofluidics in biology, chemistry, physics and engineering and the valuable knowledge available in these disciplines. First, the forces that play a role on the nanoscale are discussed and then a summary is given of some different theoretical treatments. Subsequently, an overview is given of the different phenomena occurring on the nanoscale and their present applications. Finally, some possible future applications are discussed.
An easily fabricated resizable nanometre-scale aperture provides a novel class of electrical device that can be deployed as a tunable molecular sensor. Here, we demonstrate that the metered penetration of an elastomeric membrane with a sharp probe yields an aperture that, by reversible adjustment of the membrane, provides a spectrum of geometries suitable for single particle analysis. We describe such an aperture optimised in situ for the detection and controlled gating of DNA molecules. (c) 2006 Elsevier B.V. All rights reserved.
Accurate modeling of diffusive transport of nanoparticles across nanopores is a particularly challenging problem. The reason is that for such narrow pores the large surface-to-volume ratio amplifies the relevance of the nanoscopic details and of the effective interactions at the interface with pore walls. Close to the pore wall, there is no clear separation between the length scales associated with molecular interactions, layering of the solvent at the interface with the pore and the particle size. Therefore, the standard hydrodynamic arguments may not apply and alternative solutions to determining average transport coefficients need to be developed. We here address this problem by offering a multiscale ansatz that uses effective potentials determined from molecular dynamics simulations to parametrise a four state stochastic model for the positional configuration of the particle in the pore. This is in turn combined with diffusivities in the centre of the pore and at the pore wall to calculate the average diffusion constant. We apply this model to the diffusion of fullerenes in a toluene filled slit nanopore and calculate the mean diffusion coefficient as a function of the pore size. We show that the accuracy of our model is affected by the partial slip of the toluene on the pore wall.
Extracellular vesicles (EVs) released from biological cells have attracted considerable interest due to their potential for cancer diagnostics and important role in cell signaling. Most previously reported studies have been concerned with the detection of EVs in biofluids and analysis of proteins and nucleic acids they contain. Electrochemical resistive-pulse (ERP) sensing enables direct detection of single EVs released from a specific cell and analysis of reactive oxygen and nitrogen species in such vesicles. Here, we demonstrate the applicability of ERP sensing to distinguish between nontransformed and cancerous breast cell lines as well as between breast cancer cell lines with different metastatic potential. Another application of ERP sensing is in real-time monitoring of changes in a single cell induced by a chemical agent. This approach is potentially useful for evaluating the efficacy of therapeutic agents, including those that trigger breast cancer cell death by inducing intense oxidative stress.
The use of functional DNA nanostructures as carriers to ship proteins through solid-state nanopores has recently seen substantial growth in single-protein-molecule detection (SPMD), driven by the potential of this methodology and implementations that it may enable. Ultrasmall nanopores have exhibited obvious advantages in spatiotemporal biological detection due to the appropriate nanoconfined spaces and unique properties. Herein, a 6.8 nm DNA tetrahedron (TDN) with a target-specific DNA aptamer (TDN-apt) was engineered to carry the representative target of acetylcholinesterase (AChE) through an ultrasmall nanopipet with a 30 nm orifice, underpinning the advanced SPMD of AChE with good performance in terms of high selectivity, low detection limit (0.1 fM), and especially superior signal-to-noise ratio (SNR). The kinetic interaction between TDN-apt and AChE was studied and the practical applicability of the as-developed SPMD toward real samples was validated using serum samples from patients with Alzheimer's disease. This work not only presented a feasible SPMD solution toward low-abundance proteins in complex samples and but also was envisioned to inspire more interest in the design and implementation of synergized DNA nanostructure-ultrasmall nanopore systems for future SPMD development.
Detection of extracellular vesicles (EVs) exosomes is a challenge to address the need for better diagnostic tests and to create a point-of-care (POC) platform that can detect, monitor and treat health conditions early to allow personalized therapies. A multidisciplinary approach is needed to address these health-related technical issues. Over the past decade, materials scientists and engineers have worked on the same platform to develop flexible, lightweight, miniaturized, and integrated POC devices for exosome detection. Therefore, exosome detection based on various nanomaterials is of particular interest. In this paper, we describe the current state of knowledge on 0D-3D nanostructured materials and present a POC-based technique for exosome detection. Finally, the challenges that need to be solved to expand their clinical application are discussed.
It is important to detect cancer biomarkers at an early stage of tumor development for the effective diagnosis and treatment of cancer. As a well-known probe for detecting superoxide (·O2-) radicals, nitro blue tetrazolium (NBT) can rapidly react with ·O2- to form a hydrophobic formazan precipitate. In this study, by deliberately utilizing this reaction, Pt asymmetrically decorated on a TiO2 nanochannel membrane (Pt/TiNM) is explored to fabricate an electrochemical immunosensing platform with outstanding selectivity and ultrahigh sensitivity. Using NBT as the substrate, hydrophobic formazan precipitation induces a substantial block of ionic diffusion flux in nanochannels. Using alpha fetoprotein (AFP) as the target analyte, the established immunorecognition event was used to induce MoS2-Ab2 conjugates. Thanks to the excellent light-shielding ability of MoS2 nanosheets, the production of ·O2- radicals from the photocatalysis of Pt/TiNM is effectively depressed because of the attenuated arrival of light. The reduced formazan precipitation results in ionic transport changes in nanochannels, which in turn enables the selective recognition of AFP down to 2 ng mL-1. This target-modulated sensing strategy is also capable of sensing other immune targets, thus paving a new way for designing nanochannel-based sensing platforms.
Artificial ion channels with tunable ionic transport control and intelligent iontronic functions at the nanoscale have a wide application in logic computing and biosensing. Although some artificial ion channels with smart ion transport characteristics have been developed, most of them are constructed on porous membranes with undefined channel numbers. It is still challenging to achieve multiple ion transport features in single nanochannels and to control the ion flow more accurately with excellent repeatability. In this paper, a design strategy is presented to fabricate pH-responsive ion channels with various ion transport features based on a single polydimethylsiloxane (PDMS) nanochannel. The single-ion nanochannel developed by this approach can be further integrated into electronic systems on a chip. Three types of artificial ion channels are demonstrated and investigated systematically in this work. With symmetric or asymmetric pH stimuli, these ion channels can alternatively change their working states among an opened state, a closed state, and an ionic diode state. Four different ion transport features can be realized in a triple-gated ion channel system. With these advantages of the design, it is promising to build smart nanofluidic iontronic devices with widespread applicability in energy conversions, active ion transport control, and biological analysis.
Thermoplastic nanofluidic devices are promising platforms for sensing single biomolecules due to their mass fabrication capability. When the molecules are driven electrokinetically through nanofluidic networks, surface charges play a significant role in the molecular capture and transportation, especially when the thickness of the electrical double layer is close to the dimensions of the nanostructures in the device. Here, we used multivalent cations to alter the surface charge density of thermoplastic nanofluidic devices. The surface charge alteration was done by filling the device with a multivalent ionic solution, followed by withdrawal of the solution and replacing it with KCl for conductance measurement. A systematic study was performed using ionic solutions containing Mg²⁺ and Al³⁺ for nanochannels made of three polymers: poly(ethylene glycol) diacrylate (PEGDA), poly(methyl methacrylate) (PMMA) and cyclic olefin copolymer (COC). Overall, multivalent cations within the slip plane decreased the effective surface charge density of the device surface and the reduction rate increased with the cation valency, cation concentration and the surface charge density of thermoplastic substrates. We demonstrated that a 10-nm diameter in-plane nanopore formed in COC allowed translocation of λ-DNA molecules after Al³⁺ modification, which is attributed to the deceased viscous drag force in the nanopore by the decreased surface charge density. This work provides a general method to manipulate surface charge density of nanofluidic devices for biomolecule resistive pulse sensing. Additionally, the experimental results support ion-ion correlations as the origin of charge inversion over specific chemical adsorption.
Resistive-pulse sensing with solid-state nanopores is a sensitive, label-free technique for analyzing single molecules in solution. To add functionality to resistive-pulse measurements, direct coupling of the nanopores to other pores and nanoscale fluidic elements, e.g., reactors, separators, and filters, in the same device is an important next step. One approach is monolithic fabrication of the fluidic elements in the plane of the substrate, but methods to generate pores with circular cross sections are needed to improve sensing performance with in-plane devices. Here, we report a fabrication method that directly patterns nanopores with circular cross sections in series and in plane with the substrate. A focused ion beam instrument is used to mill a lamella in a nanochannel and, subsequently, bore a nanopore through the lamella. The diameter and geometry of the nanopore are controlled by the current and dose of the ion beam and by the tilt angle and thickness of the lamella. We fabricated devices with vertical and tilted lamellae and nanopores with diameters from 40 to 90 nm in cylindrical and conical geometries. To test device performance, we conducted resistive-pulse measurements of hepatitis B virus capsids. Current pulses from T = 3 capsids (∼31 nm diameter) and T = 4 capsids (∼35 nm diameter) were well resolved and exhibited relative pulse amplitudes (Δi/i) up to 5 times higher than data obtained on nanopores with rectangular cross sections. For smaller pore diameters (<45 nm), which approach the diameters of the capsids, a dramatic increase in the pulse amplitude was observed for both T = 3 and T = 4 capsids. Two and three pores fabricated in series further improved the resolution between the relative pulse amplitude distributions for the T = 3 and T = 4 capsids by up to 2-fold.
Solid-state nanopore has the ability to detect proteins at a single-molecule level with its high sensitivity, high-throughput, and low cost. Improvements in fabrication, functionalization, and characterization of solid-state nanopores keep evolving. Various analytical methods targeted towards diagnostic applications using nanopore-based devices are appearing. This review article provides an overview of recent progress in the field of solid-state and biological nanopores for protein detection in a complex analyte. The advantages and challenges involved in nanopore sensing have been discussed. Further, the review surpasses the steady-state resistive pulse techniques of sensing and incorporate transient variations in the nanopore conductance. Application of the power spectral density of these fluctuations toward sensing has been highlighted with importance on reducing the detection limit in a complex environment. Lastly, the current problems and future perspectives have been discussed with a perspective to increase nanopores performance towards diagnostic applications in complex medium.
Detection of small biomolecules is critical for understanding molecular mechanisms in biological systems and performing in vitro diagnosis in clinics. Current antibody based detection methods face large challenges in detecting small biomolecules at low concentrations. We report a new method for detecting small biomolecules based on molecular recognition and nanoparticle (NP) counting. Aptamer-functionalized NPs are attached to complementary sequence (CS)-conjugated microparticle (MP) carriers. In the presence of target small biomolecules at ultra low concentrations, NPs would be released from the MP carriers. Coupled with a resistive pulse sensor (RPS) using a micropore that counts the released NPs, this method can measure the concentrations of target biomolecules at low concentrations with high sensitivity and high throughput. Adenosine was used as a model to demonstrate the feasibility of this method. It is demonstrated that this method can detect a wide range of adenosine concentrations with a low detection limit of 0.168 nM, which is 10 times lower than that of the ELISA kit. With its simple structure, high sensitivity, and high reproducibility, this detection method holds great potential for the ultrasensitive detection of low abundance small biomolecules.
Nanopore sequencing is currently a widely used technology for single-molecule DNA detection and analysis. Although the error rate is still too high to enable diagnostic applications, biological nanopores have demonstrated to be able to read the sequence of single DNA molecule, and massive efforts are now on-going to exploit the same technology for decoding single proteins. In this framework, solid-state nanopore-based sensors have emerged as alternative platforms for single-molecule sequencing. They did not demonstrate yet to enable reliable DNA sequencing, but they can be easily engineered to include additional functionalities, such as mechanical robustness and versatile chemical functionalization. Among the different classes of solid-state nanopores, plasmonic nanopores have rapidly attracted the interest within the nanopore community, since they allow to engineer the electromagnetic field, which can be exploited as an advanced tool for enhanced optical spectroscopy and tweezing, as well as local control over temperature. More importantly, plasmonic nanopores are the most versatile platform to realize electro-optical single-molecule detection and to explore integrated sequencing methods. In particular, the use of optical read-out in single-molecule sequencing is a key aspect to tackle the challenging goal of protein sequencing, a goal that is more than challenging to achieve using electrical read-out approaches. This critical Review offers a comprehensive understanding of the most recent state-of-the-art plasmonic nanopores for single-molecule detection and biomolecule sequencing applications, and discusses the latest advances and future perspectives on plasmonic nanopore-based technologies, focusing the discussion on the critical aspects to be overcome to enable multiplexing analysis of DNA and protein with single-molecule sensitivity.
A full investigation of DNA translocation though a surface-modified nanopore was performed. The effect of tip diameter and their average duration and current-pulse amplitudes were reported. Finally, the current-time traces taken under relatively low-electrolyte concentration was also studied and compared with higher concentrations.
Abstract
The ability to detect DNA strands and determine their characteristics in solution plays a critical role to design a sensor in terms of its biophysical properties and receives attention in whole- genome sequencing. In this study, we report a simple method to detect 100-bp DNA based on resistive-pulse sensing and its characteristics in terms of analyte concentration, potential, tip size. and electrolyte concentration. Track-etched polyethyleneterephtalate (PET) nanopores were used and modified (w/EDC-NHS coupling) to decrease the negative surface charge and promote DNA translocation. We tuned up the tip diameters of nanopores by using symmetric and asymmetric chemical etching. The tip diameters ranged from 21 nm to 42 nm. To show the electrophoretic nature of translocation, we investigated the concentration and potential dependence. The current-pulse events were observed down to 600 mV (4±1 events/min) and 0.25 nM was the minimum concentration. Both potential and concentration dependence showed linear behavior which agreed to previous studies. The effect of tip size was also studied and its effect on translocation frequency was discussed. Finally, the change in current-pulse amplitude and duration as a function of electrolyte concentration was studied and longer duration values (14.3±1.4 ms) were observed. Our findings provide a guide to translocate the charged molecules more efficiently and help to understand the translocation dynamics of short DNA molecules in resistive-pulse sensing.
Controlling and patterning the surface charges of the sensing nanochannel in a resistive pulse sensing (RPS) device to enhance the signal detection are investigated in this paper. The electric currents and signal detection in the sensing nanochannels with positive surface charges, negative surface charges, and opposite surface charges at the two ends are systematically studied. It is found that the length of the surface-modified nanochannels has a significant effect on the sensitivity of the RPS detection. The pulse signal amplitude can be increased by 3 times in the surface-modified nanochannel when the channel length is 500 nm. The enhancement in the signal amplitude becomes less prominent in long nanochannels. Modified sensing nanochannels with a channel length of 5μm are fabricated by regulating surface charges with charged polyelectrolytes. Nanoparticles with a diameter of 5 nm are detected in the modified nanochannels. The experimental results are in good agreement with the numerical simulation results. It is also found that the surface charge pattern of the sensing nanochannel has a significant effect on the sensitivity of the RPS detection. The RPS signals can be enhanced by approximately 50% when the sensing nanochannel has a section with positive surface charge and negative surface charge in the rest channel in a KCl solution with a bulk concentration of 100 mM.
In this paper, metal-insulator-metal based plasmonic sensor with a straight waveguide and a side coupled equilateral triangular resonator (disc-type and ring-type) has been proposed and analyzed. The detection of refractive index change has been numerically simulated and analyzed using COMSOL Multiphysics. The sensitivity achieved using our proposed structure has been found to be 2713 nm/RIU with a high figure of merit (FOM) of 35.1. It is shown that the sensor can detect a refractive index change of 3.68×10⁻⁶, which is the sensing resolution of the sensor, for dielectrics whose refractive indices are between 1 to 1.03 and also between 1.33 to 1.42. Simplicity and compact design with high refractive index sensitivity and FOM of the plasmonic device helps to design bio-sensing devices, in biomedical applications like cancer cell detection, nano-sensing devices and also optical filter applications.
We have developed a new three-dimensional (3D) surface for use in biosensors that is based on modified novel thorns-like polyelectrolytes (3D-PETx), which comprises of poly-l-lysine (PLL) appended with multitude oligo (ethylene glycol) (OEG) and biotin moieties. It tethered to the sensor surface by PLL, while the OEG-biotin chains are forced to stretch away from the surface for target detections. Due to its 3D structure, the number of the OEG-biotin per surface unit is markedly increased compared to conventional 2D polyelectrolytes (2D-PET) coating. Their antifouling property and sensing performance for human IgG and PSA were compared with the 2D-PET by BioLayer Interferometry (Blitz), Surface Plasmon Resonance (SPR), microfluidic devices and Enzyme-Linked ImmunoSorbent Assay (ELISA). Experimental results show that 3D-PETx presents higher sensitivity for biomarker detection both in buffer and in serum and provides an almost non-fouling surface even in undiluted serum. In addition, a sensitive PSA detection was achieved in undiluted serum with a LOD down to 0.6 ng/mL. The successful immunosensing in undiluted serum demonstrate the potential of the 3D-PETx coating for real clinical applications.
Objective
The aim of this study is to review the current evidence on the vertical transmission of SARS CoV-2.
Methods
Combination of the following keywords; COVID-19, SARS CoV-2, placenta, vertical transmission, intrauterine infection, breast milk were searched in databases.
Results
In the 50 studies included, 17 newborns testing positive for SARS CoV-2 by RT-PCR were reported. In three neonates, SARS-CoV-2 IgG and IgM levels were elevated. Eight placental tissues testing positive for the virus were reported. Three positive RT-PCR results of test of breast milk have been reported recently. One amniotic fluid testing positive was reported.
Conclusion
Possible vertical transmission of SARS CoV-2 has been observed in some studies currently. More RT-PCR tests on amniotic fluid, placenta, breast milk and cord blood are required.
Since exosomes carry the genetic information of their parental cells, there is an increasing need for new technologies capable of detecting exosomes with high sensitivity and accuracy. In this study, we propose a dual antibody-modified nanochannel biosensor for rapid, sensitive analysis of exosomes. Two antibodies (anti-CD63 and anti-EpCAM) were simultaneously immobilized on the surface of the porous anodic alumina (PAA) nanochannel to combine with proteins overexpressed on the exosomal membrane. As such, exosomes could be efficiently captured by the dual antibody-modified nanochannel. The captured exosomes effectively covered the nanochannel inlet, leading to remarkable block of ion flow through the channel. Based on this principle, the captured exosomes could be sensitively and accurately detected using a picoammeter. It was found that the use of two antibodies greatly increased the capture efficiency of exosomes compared to that of a single antibody. The results showed that exosomes down to 7.3 × 10³ particles/mL were successfully detected by the nanochannel. Moreover, exosomes were successfully detected from plasma samples using a nanochannel biosensor modified with dual antibody, and the results showed significant differences in the exosomes content in plasma samples of cancer patients and healthy people, so as to initially distinguish cancer patients from healthy people.
The modification of cylindrical anodic aluminum oxide (AAO) nanopores by alternating layer by layer (LBL) deposition of poly(sodium-4-styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) was studied in situ by Reflectometric Interference Spectroscopy (RIfS). In particular, the kinetics of polyelectrolyte deposition inside the 37 ± 3 nm diameter and 3.7 ± 0.2 µm long pores was unraveled and potential differences in LBL multilayer growth compared to flat silicon substrates and the effect of different ionic strengths and different types of ions were investigated. RIfS measures the effective optical thicknesses, which is for constant pore length proportional to the effective refractive index of the AAO sample, from which in turn the deposited mass of polymer or the corresponding layer thickness can be estimated. Compared to the multilayer growth by LBL deposition on flat amino-silane primed silicon wafers, which was assessed by spectroscopic ellipsometry, the thickness increment per deposited bilayer as well as the dependence of this increment on ionic strength (0.01 to 0.15) and counter ion type (Na+ vs. Ca2+) inside the amino-silane primed nanopores was for the first bilayers to within the experimental error identical. For thicker multilayers, the pore diameter became smaller, which led to reduced thickness increments and eventually virtually completely filled pores. The observed kinetics is consistent with a mass transport limited adsorption of the polyelectrolyte to the charged surface according to a Langmuir isotherm with negligible desorption rate. In addition to fundamental insight into the build-up of polyelectrolyte multilayers inside AAO nanopores, our results highlight the sensitivity of RIfS and its use as analytical tool for probing processes inside nanopores and for the development of biosensors.
Detection and characterization of individual nanoparticles less than 100 nm are important for semiconductor manufacturing, environmental monitoring, biomedical diagnostics, and drug delivery. Photothermal spectroscopy is a light absorptiometry and promising method for detection and characterization because of its high sensitivity and selectivity compared with light scattering or electrical detection methods. However, the characterization of individual nanoparticles in liquids is still challenging for conventional photothermal detection methods. Here, we report a method for the ultrasensitive detection and accurate characterization of individual nanoparticles in liquids by photothermal optical diffraction, which utilizes enhancement of optical diffraction by a nanochannel after light absorption and heat generation of individual nanoparticles in the channel. Our method realized individual 20 nm Au nanoparticle detection with almost 100% detection efficiency by utilizing nanochannels, leading to concentration determination without a calibration curve. Furthermore, we measured individual nanoparticle size and discriminated 20 and 40 nm Au nanoparticles from their photothermal signals. Our photothermal-based nanoparticle detection method in nanochannels has a potential for a wide range of applications such as on-site evaluation of synthesized plasmonic nanoparticles and drug delivery particles.
Microfluidics has achieved integration of chemical processing in microspaces and realized miniaturized analyses in the fields such as chemistry and biology. We have proposed a general concept of integration and extended this concept to the 10-1000 nm space exploring ultimate analytical performances (e.g. immunoassay of a single-protein molecule). However, a sampling method is still challenging for nanofluidics despite its importance in analytical chemistry. In this study, we developed a femtoliter (fL) sampling method for volume measurement and sample transport. Traditionally, sampling has been performed using a volumetric pipette and flask. In this research, a nanofluidic device consisting of a femtoliter volumetric pipette and flask was fabricated in glass substrates. Since gravity, which is exploited in bulk fluidic operations, becomes less dominant than surface effects on the nanometer scale, fluidic operation of the femtoliter sampling was designed based on utilizing surface tension and air pressure control. The working principle of an 11 fL volumetric pipette and a 50 fL flask, which were connected by a nanochannel, was verified. It was found that evaporation of the sample solution due to air flow was a significant source of error because of the ultra-small volumes being processed. Thus, the evaporation issue was solved by suppressing air flow. As a result, the volumetric measurement error was decreased to ±0.06 fL (CV 0.6%), which is sufficiently low for using in nanofluidic analytical applications. This study will be a fundamental technology for the development of novel analytical methods for femtoliter volume samples such as single molecule analyses.
Nanopores as artificial biomimetic nanodevices are of great importance for their applications in biosensing, nanomedicine and bioelectronics. However, it remains a challenge to detect small biomolecules especially small-sized proteins with high sensitivity and selectivity. In the article, we report a simple and efficient method for small-sized protein detection by constructing biphasic-pulse nanopore biosensor. Unlike the traditional resistive pulse sensing, the biphasic-pulse event can provide unique and abundant fingerprint information. Although the nanopore biphasic-pulse electrical signal is originated from both the molecular exclusion electrical resistance and the surface-charged effect of confined molecule, its frequency and amplitude of the waveform can be adjusted by pH, applied potential and salt concentration. Based on the frequency of the biphasic pulse, nanomolar concentration of proteins could be specifically detected and the limit of detection is 1.2 nM. In addition, the biphasic-pulse nanopore shows well discrimination in similar-sized protein detection and its signal generation is highly reproducible. The nanopore biphasic-pulse biosensor should have broad applications as a new generation of powerful single-molecule device.
In this work, we propose a novel methodology for the electrical monitoring using nanoporous alumina membranes of virulence factors secreted by bacterial pathogens. Bacterial hyaluronidase (HYAL), which is produced by a number of invasive Gram-positive bacteria, is selected as a model compound to prove the concept. Our electrochemical set-up takes advantage of the flat surface of ITO/PET electrodes for their assembling with the nanoporous membrane. The proposed analytical method, based on the electrical monitoring of the steric/electrostatic nanochannels blocking upon formation of an antibody-HYAL immunocomplex, reached detection limits as low as 64 UI/mL (17.3 U/mg) HYAL. The inert surface of the ITO/PET electrodes together with the anti-biofilm properties of the 20 nm-pore sized alumina membranes allow for culturing the bacteria, capturing the secreted enzymes inside the nanochannels and removing the cells before the electrochemical measurement. Secreted HYAL at levels of 1000 UI/mL (270 U/mg) are estimated in Gram-positive S. aureus cultures, while low levels are detected for Gram-negative P. aeruginosa (used as a negative control). Finally, HYAL secretion inhibition by RNAIII-inhibiting peptide (YSPWTNF-NH2) is also monitored, opening the way to further applications of the developed monitoring system for evaluation of the anti-virulence potential of different compounds. This label-free method is rapid and cheap, avoiding the use of the time consuming sandwich assays. We envisage future applications for monitoring of bacterial virulence/invasion as well as for testing of novel antimicrobial/anti-virulence agents.
Single protein sensing based on solid-state nanopore is promising but challenging because the fast translocation velocity of a protein is beyond the bandwidth of nanopore instruments. To decelerate the translocation speed, here, we employed a common protein crosslink interaction to achieve a general and robust nanopore sensing platform for single-molecule detection of protein. Benefiting from the EDC/NHS coupling interaction between nanopore and pro-teins, a ten-fold decrease in speed has been achieved. The clearly distinguishable current signatures further reveal the anisotropy of a protein produces three translocation behav-iors, which are horizontal, vertical and flipping transit inside nanopore confinement. This strategy provides a general platform for rapid detection of proteins as well as exploring fundamental protein dynamics at the single-molecule level.
Tunable resistive pulse sensing (TRPS) uses the Coulter principle to detect, measure and analyse particles at length scales ranging from tens of nanometres through to microns. The technology and its associated methods have advanced so that TRPS is regularly used as a characterization technique in peer-reviewed studies. This Perspective is concerned with opportunities to further develop TRPS, with a specific focus on improved measurement of size and charge for submicron particles. There is currently broad demand for increased rigor in such measurements. Particular points of interest include consistent use of statistics, development of accurate physical models, and realistic assessment of uncertainties associated with the usual measurement protocols. Highlights from recent studies involving TRPS are also reviewed. The technique is particularly popular in the burgeoning research field relating to extracellular vesicles, and the range of biologically relevant applications also includes liposomes, viruses, and on-bead assays.
This paper reports a surface functionalization strategy for protein detections based on biotin-derivatized poly(L-lysine)-grafted oligo-ethylene glycol (PLL-g-OEGx-Biotin) copolymers. Such strategy can be used to attach the biomolecule receptors in a reproducible way simply by incubation of the transducer element in a solution containing such copolymers which largely facilitated the sensor functionalization at an industrial scale. As the synthesized copolymers are cationic in physiology pH, surface biotinylation can be easily achieved via electrostatic adsorption on negatively charged sensor surface. Biotinylated receptors can be subsequently attached through well-defined biotin-streptavidin interaction. In this work, the bioactive sensor surfaces were applied for mouse IgG and prostate specific antigen (PSA) detections using quartz crystal microbalance (QCM), optical sensor (BioLayer Interferometry) and conventional ELISA test (colorimetry). A limit of detection (LOD) of 0.5 nM was achieved for PSA detections both in HEPES buffer and serum dilutions in ELISA tests. The synthesized PLL-g-OEGx-Biotin copolymers with different OEG chain length were also compared for their biosensing performance. Moreover, the surface regeneration was achieved by pH stimulation to remove the copolymers and the bonded analytes, while maintaining the sensor reusability as well. Thus, the developed PLL-g-OEGx-Biotin surface assembling strategy is believed to be a versatile surface coating method for protein detections with multi-sensor compatibility.
Artificial nanochannels, inheriting smart gating functions of biological ion channels, promote the development of artificial functional nanofluidic devices for high-performance biosensing and electricity generation. However, gating states of the artificial nanochannels have been mainly realized through chemical modification of the channels with responsive molecules and their gating states cannot be further regulated once the nanochannel is modified. In this work, we employed a new supramolecular layer-by-layer (LbL) self-assembly method to achieve reversible and adjustable multiple gating features in nanofluidic diodes. Initially, a self-assembly precursor was modified into a single conical nanochannel, then host molecule-cucurbit[8]uril (CB[8]) and guest molecule, a naphthalene derivative, were self-assembled onto the precursor through LbL method driven by host-enhanced π-π interaction, forming supramolecular monolayer or multilayers on the inner surface of the channel. These self-assemblies with different layer numbers possessed remarkable charge effects and steric effects, exhibiting a capability to regulate the surface charge density and polarity, the effective diameter and the geometric asymmetry of the single nanochannel, realizing reversible gating of the single nanochannel among multiple rectification and ion-conduction states. As an example of self-assembly of supramolecular networks in nano-confinements, this work provides a new approach for enhancing functionalities of artificial nanochannels by LbL supramolecular self-assemblies. Meanwhile, since the host molecule, CB[8], used in this work can interact with different kinds of biomolecules and stimuli-responsive chemical species, this work can be further extended to build a novel stable multiple-state research platform for a variety of uses such as sensing and controllable release.
A new surface functionalization scheme for nano-Bio field effect transistors (FETs) using biocompatible polyelectrolyte thin films (PET) is developed. PET assemblies on Si nanowires (Si-NWs) are driven by electrostatic interactions between the positively charged polymer backbone and negatively charged Si/SiO2 surface. Such assemblies can be directly coated from PET aqueous solutions and result in a uniform nanoscale thin film, which is more stable compared to the conventional amine silanization. Short oligo-ethylene glycol chains are grafted on the PETs to prevent nonspecific protein binding. Moreover, the reactive groups of the polymer chains can be further functionalized to other chemical groups in specific stoichiometry for biomolecules detection. Therefore, it opens a new strategy to precisely control the functional group densities on various biosensor surfaces at the molecular level. In addition, such assemblies of the polymers together with the bound analytes can be removed with the pH stimulation resulting in regeneration of a bare sensor surface without compromising the integrity and performance of the Si-NWs. Thus, it is believed that the developed PET coating and sensing systems on Si-NW FETs represent a versatile, promising approach for regenerative biosensors which can be applied to other biosensors and will benefit real device applications, enhancing sensor lifetime, reliability, and repeatability.
Tunable resistive pulse sensing (TRPS) is an experimental technique that has been used to study and characterise colloidal particles ranging from approximately 50 nm in diameter up to the size of cells. The primary aim of this Review is to provide a guide to the characteristics and roles of TRPS in recent applied research. Relevant studies reflect both the maturation of the technique and the growing importance of submicron colloids in fields such as nanomedicine and biotechnology. TRPS analysis of extracellular vesicles is expanding particularly swiftly, while TRPS studies also extend to on-bead assays using DNA and aptamers, drug delivery particles, viruses and bacteria, food and beverages, and superparamagnetic beads. General protocols for TRPS measurement of particle size, concentration and charge have been developed, and a summary of TRPS technology and associated analysis techniques is included in this Review.
Elastomeric microvalves in poly(dimethylsiloxane) (PDMS) devices are today's paradigm for massively parallel microfluidic operations. Here, we report that such valves can act as nanochannels upon closure. When tuning nanospace heights between ~55 nm and ~7 nm, the nanofluidic phenomenon of concentration polarization could be induced. A wide range of concentration polarization regimes (anodic and cathodic analyte focusing and stacking) was achieved simply by valve pressure actuation. Electro-osmotic flow generated a counterpressure which also could be used to actuate between concentration polarization regimes. 1000-fold preconcentration of fluorescein was achieved in just 100 s in the anodic focusing regime. After valve opening, a concentrated sample plug could be transported through the valve, though at the cost of some defocusing. Reversible nanochannels open new avenues for integrating electrokinetic operations and assays in large scale integrated microfluidics.
A review is made, with the aim of unifying various apparently disparate sensing strategies. The unifying feature is the underlying measurement principle that entails occlusion of an aperture through which a current is passing by the analyte species. The review focuses on two very recent manifestations of this sensing paradigm: the use of protein-based channels and nanotube membranes for small molecule and ion sensing.
Layer-by-layer (LBL) films of a semiconducting polymer (POMA) alternated with a polyelectrolyte (PVS), adsorbed onto silicon oxide, mica, ITO/glass, Au/Cr/glass, hydrophilic and hydrophobic glass were studied by atomic force microscopy (AFM). The samples were characterized LBL with the AFM operating in the contact, friction and tapping modes, which allowed us to determine their morphological surface properties such as roughness, mean grain size, grain boundaries and power spectrum density. Their film thickness was measured by AFM using the tip as a scraping tool. Surface roughness increases with the number of bilayers until a constant value is reached. This is in agreement with the observed increase in the adsorbed amount (per layer) of POMA as the number of bilayers is increased, which also saturates after several bilayers. It is shown that the 3D growth behaviour indicates a similar microscopic mechanism for all systems under study, pointing to a stochastic growth process of the Eden model type, but strongly influenced by initial roughness and water affinity of the virgin substrates. The crystalline or amorphous nature of the substrates does not seem to influence the growth process.
Scanning ion occlusion sensing (SIOS), a technique that uses a tunable pore to detect the passage of individual nano-scale objects, is applied here for the rapid, accurate and direct measurement of synthetic and biological nanoparticle concentrations. SIOS is able to characterize smaller particles than other direct count techniques such as flow cytometry or Coulter counters, and the direct count avoids approximations such as those necessary for turbidity measurements. Measurements in a model system of 210-710 nm diameter polystyrene particles demonstrate that the event frequency scales linearly with applied pressure and concentration, and that measured concentrations are independent of particle type and size. Both an external-calibration and a calibration-free measurement method are demonstrated. SIOS is then applied to measure concentrations of Baculovirus occlusion bodies, with a diameter of ~1 μm, and the marine photosynthetic cyanobacterium Prochlorococcus, with a diameter of ~600 nm. The determined concentrations agree well with results from counting with microscopy (a 17% difference between the mean concentrations) and flow cytometry (6% difference between the mean concentrations), respectively.
Molecular design of ionic current rectifiers created on the basis of single conical nanopores is receiving increasing attention by the scientific community. Part of the appeal of this topic relies on the interest in sensors and fluidic nanoactuators based on the transport of ions and molecules through nanopore architectures that can readily be integrated into functional systems. The chemical modification of the pore walls controls not only the diameter of these nanoarchitectures but also their selectivity and transport properties. In order to confer selectivity to solid-state nanopores, it is necessary to develop and explore new methods for functionalizing the pore walls. Hence, the creation of functional nanopores capable of acting as selective ion channels or smart nanofluidic sensors depends critically on our ability to assemble and build up molecular architectures in a predictable manner within confined geometries with dimensions comparable to the size of the building blocks themselves. In this context, layer-by-layer deposition of polyelectrolytes offers a straightforward process for creating nanoscopic supramolecular assemblies displaying a wide variety of functional features. In this work, we describe for the first time the integration of layer-by-layer polyelectrolyte assemblies into single conical nanopores in order to study and explore the functional features arising from the creation of charged supramolecular assemblies within the constrained geometry of the nanofluidic device. To address this challenging topic, we used a combined experimental and theoretical approach to elucidate and quantify the electrostatic changes taking place inside the nanopore during the supramolecular assembly process. The multilayered films were built up through consecutive layer-by-layer adsorption of poly(allylamine hydrochloride) (PAH) and poly(styrenesulfonate) (PSS) on the pore surface. Our results show that the charge transport properties of single conical nanopores functionalized with PAH/PSS assemblies are highly dependent on the number of layers assembled on the pore wall. In contrast to what happens with PAH/PSS films deposited on planar surfaces (quantitative charge reversal), the surface charge of the pore walls decreases dramatically with the number of PAH/PSS layers assembled into the nanopore. This behavior was attributed to the nanoconfinement-induced structural reorganization of the polyelectrolyte layers, leading to the efficient formation of ion pairs and promoting a marked decrease in the net fixed charges on the nanopore walls. We consider that these results are of paramount relevance for the modification of nanopores, nanopipets, and nanoelectrodes using charged supramolecular assemblies, as well as of importance in "soft nanotechnology" provided that structural complexity, induced by nanoconfinement, can define the functional properties of self-assembled polymeric nanostructures.
Means for Counting Particles Suspended in A Fluid
- W H Coulter
W.H. Coulter, Means for Counting Particles Suspended in A Fluid, U.S. Patent.,
1953, 2656508 A.