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Resistive Pulse Sensing Device with Embedded Nanochannel (nanochannel-RPS) for Label-free Biomolecule and Bionanoparticle Analysis
Abstract
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.
... Therefore, the particle counting and sizing can be achieved by analyzing RPS signals. In our previous work, a nano-RPS device which was composed of microchannels bridged by a nanochannel on polydimethylsiloxane (PDMS), and the SiO 2 substrate patterned with microelectrodes was developed to analysis nanoparticles [40]. However, the geometric dimensions of the sensing gate are fixed once the nano-RPS are made, which limits the size range of the target analytes. ...
... The PDMS channel layer was fabricated in poly (dimethyl-siloxane) (PDMS) using soft lithography. The PDMS mold was fabricated by a multilayer photolithography process, which was described in our previous work [40]. ...
... In this work, the MS strategy was introduced to tune the PDMS-based nano-RPS device, which was proposed in our previous work [40]. And, the improvements on sensing performance of the MS-tuned nano-RPS device will be discussed systematically. ...
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.
... Additionally, resistive sensing can be used in aqueous solutions to investigate the fluid composition. There is a recent study on a resistive pulse sensing device with nanochannels embedded using nanotechnology to enable label-free biomolecule and bionanoparticle analysis, showing a promising sensing strategy that is not only capable of label-free analysis for nanoscale biomolecules and bionanoparticles but also cost-effective for large-scale manufacturing [53]. Recently, using solid-state nanopores, the dynamics of knots in doublestranded DNA under unique regimes of nanometer-scale confinement, large forces, and short time scales were studied in order to investigate the static and dynamic properties of biopolymers [54]. ...
Micro- and nanotechnology-enabled sensors have made remarkable advancements in the fields of biomedicine and the environment, enabling the sensitive and selective detection and quantification of diverse analytes. In biomedicine, these sensors have facilitated disease diagnosis, drug discovery, and point-of-care devices. In environmental monitoring, they have played a crucial role in assessing air, water, and soil quality, as well as ensured food safety. Despite notable progress, numerous challenges persist. This review article addresses recent developments in micro- and nanotechnology-enabled sensors for biomedical and environmental challenges, focusing on enhancing basic sensing techniques through micro/nanotechnology. Additionally, it explores the applications of these sensors in addressing current challenges in both biomedical and environmental domains. The article concludes by emphasizing the need for further research to expand the detection capabilities of sensors/devices, enhance sensitivity and selectivity, integrate wireless communication and energy-harvesting technologies, and optimize sample preparation, material selection, and automated components for sensor design, fabrication, and characterization.
... In principle, scaling down the dimensions of the microfluidic channel to submicrometer levels could be a solution for improving their ma nipulation ability. For example, nanofluidic devices (28,29), such as nanoscale deterministic lateral displacement (30), have been used to detect and manipulate sub100nm bioparticles. However, the en hanced interactions between the nanoparticles in such limited space induce uncontrolled agglomeration, adhesion, and even damage to the bioparticles. ...
The transport, enrichment, and purification of nanoparticles are fundamental activities in the fields of biology, chemistry, material science, and medicine. Here, we demonstrate an approach for manipulating nanospecimens in which a virtual channel with a diameter that can be spontaneously self-adjusted from dozens to a few micrometers based on the concentration of samples is formed by acoustic waves and streams that are triggered and stabilized by a gigahertz bulk acoustic resonator and microfluidics, respectively. By combining a specially designed arc-shaped resonator and lateral flow, the in situ enrichment, focusing, displacement, and continuous size-based separation of nanoparticles were achieved, with the ability to capture 30-nm polystyrene nanoparticles and continuously focus 150-nm polystyrene nanoparticles. Furthermore, exosome separation was also demonstrated. This technology overcomes the limitation of continuously manipulating particles under 200 nm and has the potential to be useful for a wide range of applications in chemistry, life sciences, and medicine.
DC electric field between two microwires in a straight microchannel was employed to electrophoretically accumulate and detect microplastics in water.
Extracellular vesicles (EVs) are nanoscale membrane vesicles which contain a wide variety of cargo such as proteins, miRNAs and lipids. A growing body of evidence suggests that EVs are promising biomarkers for disease diagnosis and therapeutic strategies. Although the excellent clinical value, their use in personalized healthcare practice is not yet feasible due to their highly heterogeneous nature. Taking the difficulty of isolation and the small size of EVs into account, the characterization of EVs at a single-particle level is both imperative and challenging. In a bid to address this critical point, more research has been directed into a microfluidic platform because of its inherent advantages in sensitivity, specificity and throughput. This review discusses the biogenesis and heterogeneity of EVs and takes a broad view of state-of-the-art advances in microfluidics-based EV research, including not only EV separation, but also the single EV characterization of biophysical detection and biochemical analysis. To highlight the advantages of microfluidic techniques, conventional technologies are included for comparison. The current status of artificial intelligence (AI) for single EV characterization is then presented. Furthermore, the challenges and prospects of microfluidics and its combination with AI applications in single EV characterization are also discussed. In the foreseeable future, recent breakthroughs in microfluidic platforms are expected to pave the way for single EV analysis and improve applications for precision medicine.
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Tumor-derived extracellular vesicles (EVs) present in bodily fluids are emerging liquid biopsy markers for non-invasive cancer diagnosis and treatment monitoring. Since the majority of EVs in circulation are not of tumor origin, it is critical to develop new platforms capable of enriching tumor-derived EVs from the blood. Herein, we introduce a biostructure-inspired NanoVilli Chip, capable of highly efficient and reproducible immunoaffinity capture of tumor-derived EVs from blood plasma samples. Anti-EpCAM-grafted silicon nanowire arrays were engineered to mimic the distinctive structures of intestinal microvilli, dramatically increasing surface area and enhancing tumor-derived EV capture. RNA in the captured EVs can be recovered for downstream molecular analyses by reverse transcription Droplet Digital™ PCR. We demonstrate that this assay can be applied to monitor the dynamic changes of ROS1 rearrangements and epidermal growth factor receptor T790M mutations that predict treatment responses and disease progression in non-small cell lung cancer patients.
Complete blood cell counts (CBCs) are one of the most commonly ordered and informative blood tests in hospitals. The results from a CBC, which typically include white blood cell (WBC) counts with differentials, red blood cell (RBC) counts, platelet counts and hemoglobin measurements, can have implications for the diagnosis and screening of hundreds of diseases and treatments. Bulky and expensive hematology analyzers are currently used as a gold standard for acquiring CBCs. For nearly all CBCs performed today, the patient must travel to either a hospital with a large laboratory or to a centralized lab testing facility. There is a tremendous need for an automated, portable point-of-care blood cell counter that could yield results in a matter of minutes from a drop of blood without any trained professionals to operate the instrument. We have developed microfluidic biochips capable of a partial CBC using only a drop of whole blood. Total leukocyte and their 3-part differential count are obtained from 10 μL of blood after on-chip lysing of the RBCs and counting of the leukocytes electrically using microfabricated platinum electrodes. For RBCs and platelets, 1 μL of whole blood is diluted with PBS on-chip and the cells are counted electrically. The total time for measurement is under 20 minutes. We demonstrate a high correlation of blood cell counts compared to results acquired with a commercial hematology analyzer. This technology could potentially have tremendous applications in hospitals at the bedside, private clinics, retail clinics and the developing world.
Resistive-pulse sensing has generated considerable interest as a technique for characterizing nanoparticle suspensions. The size, charge, and shape of individual particles can be estimated from features of the resistive pulse, but the technique suffers from an inherent variability due to the stochastic nature of particles translocating through a small orifice or channel. Here we report a method, and associated automated instrumentation, that allows repeated pressure-driven translocation of individual particles back and forth across the orifice of a conical nanopore, greatly reducing uncertainty in particle size that results from streamline path distributions, particle diffusion, particle asphericity, and electronic noise. We demonstrate ~0.3 nm resolution in measuring the size of nominally 30- and 60-nm radius Au nanoparticles of spherical geometry; Au nanoparticles in solution that differ by ~1 nm in radius are readily distinguished. The repetitive translocation method also allows differentiating particles based on surface charge density, and provides insights into factors that determine the distribution of measured particle sizes.
Nanopores can probe the structure of biopolymers in solution; however, diffusion makes it difficult to study the same molecule for extended periods. Here we report devices that entropically trap single DNA molecules in a 6.2-femtolitre cage near a solid-state nanopore. We electrophoretically inject DNA molecules into the cage through the nanopore, pause for preset times and then drive the DNA back out through the nanopore. The saturating recapture time and high recapture probability after long pauses, their agreement with a convection-diffusion model and the observation of trapped DNA under fluorescence microscopy all confirm that the cage stably traps DNA. Meanwhile, the cages have 200 nm openings that make them permeable to small molecules, like the restriction endonuclease we use to sequence-specifically cut trapped DNA into fragments whose number and sizes are analysed upon exiting through the nanopore. Entropic cages thus serve as reactors for chemically modifying single DNA molecules.
Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach.
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).
Particle counting in microfluidic devices with coulter principle finds many applications in health and medicine. Cell enumeration using microfluidic particle counters is fast and requires small volumes of sample, and is being used for disease diagnostics in humans and animals. A complete characterization of the cell counting process is critical for accurate cell counting especially in complex systems with samples of heterogeneous population interacting with different reagents in a microfluidic device. In this paper, we have characterized the electrical cell counting process using a microfluidic impedance cytometer. Erythrocytes were lysed on-chip from whole blood and the lysing was quenched to preserve leukocytes which subsequently pass through a 15 μm × 15 μm measurement channel used to electrically count the cells. We show that cell counting over time is a non-homogeneous Poisson process and that the electrical cell counts over time show the log-normal distribution, whose skewness can be attributed to diffusion of cells in the buffer that is used to meter the blood. We further found that the heterogeneous cell population (i.e. different cell types) shows different diffusion characteristics based on the cell size. Lymphocytes spatially diffuse more as compared to granulocytes and monocytes. The time difference between the cell occurrences follows an exponential distribution and when plotted over time verifies the cell diffusion characteristics. We also characterized the probability of occurrence of more than one cell at the counter within specified time intervals using Poisson counting statistics. For high cell concentration samples, we also derived the required sample dilution based on our particle counting characterization. Buffer characterization by considering the size based particle diffusion and estimating the required dilution are critical parameters for accurate counting results.
To date, solid-state nanopores have been fabricated primarily through a focused-electronic beam via TEM. For mass production, however, a TEM beam is not suitable and an alternative fabrication method is required. Recently, a simple method for fabricating solid-state nanopores was reported by Kwok, H. et al. and used to fabricate a nanopore (down to 2 nm in size) in a membrane via dielectric breakdown. In the present study, to fabricate smaller nanopores stably-specifically with a diameter of 1 to 2 nm (which is an essential size for identifying each nucleotide)-via dielectric breakdown, a technique called "multilevel pulse-voltage injection" (MPVI) is proposed and evaluated. MPVI can generate nanopores with diameters of sub-1 nm in a 10-nm-thick Si3N4 membrane with a probability of 90%. The generated nanopores can be widened to the desired size (as high as 3 nm in diameter) with sub-nanometre precision, and the mean effective thickness of the fabricated nanopores was 3.7 nm.
Microfluidic devices based on the Coulter principle require a small aperture for cell counting. For applications using such cell counting devices, the volume of the sample also needs to be metered to determine the absolute cell count in a specific volume. Hence, integrated methods to characterize and meter the volume of a fluid are required in these microfluidic devices. Here, we present fluid flow characterization and electrically-based sample metering results of blood through a measurement channel with a cross-section of 15 μm × 15 μm (i.e. the Coulter aperture). Red blood cells in whole blood are lysed and the remaining fluid, consisting of leukocytes, erythrocyte cell lysate and various reagents, is flown at different flow rates through the measurement aperture. The change in impedance across the electrodes embedded in the counting channel shows a linear relationship with the increase in the fluid flow rate. We also show that the fluid volume can be determined by measuring the decrease in pulse width, and increase in number of cells as they pass through the counting channel per unit time.
Roughly 33 million people worldwide are infected with HIV; disease burden is highest in resource-limited settings. One important diagnostic in HIV disease management is the absolute count of lymphocytes expressing the CD4(+) and CD8(+) receptors. The current diagnostic instruments and procedures require expensive equipment and trained technicians. In response, we have developed microfluidic biochips that count CD4(+) and CD8(+) lymphocytes in whole blood samples, without the need for off-chip sample preparation. The device is based on differential electrical counting and relies on five on-chip modules that, in sequence, chemically lyses erythrocytes, quenches lysis to preserve leukocytes, enumerates cells electrically, depletes the target cells (CD4 or CD8) with antibodies, and enumerates the remaining cells electrically. We demonstrate application of this chip using blood from healthy and HIV-infected subjects. Erythrocyte lysis and quenching durations were optimized to create pure leukocyte populations in less than 1 min. Target cell depletion was accomplished through shear stress-based immunocapture, using antibody-coated microposts to increase the contact surface area and enhance depletion efficiency. With the differential electrical counting method, device-based CD4(+) and CD8(+) T cell counts closely matched control counts obtained from flow cytometry, over a dynamic range of 40 to 1000 cells/μl. By providing accurate cell counts in less than 20 min, from samples obtained from one drop of whole blood, this approach has the potential to be realized as a handheld, battery-powered instrument that would deliver simple HIV diagnostics to patients anywhere in the world, regardless of geography or socioeconomic status.
Today, droplet based microfluidics has become a standard platform for high-throughput single cell experimentation and analysis. However, until now no label-free, integrated single cell detection and discrimination method in droplets is available. We present here a microfluidic chip for fast (>100 Hz) and label-free electrical impedance based detection of cells in droplets. The microfluidic glass-PDMS device consists of two main components, the droplet generator and the impedance sensor. The planar electrode pair in the main channel allows the detection of only cells and cell containing droplets passing the electrodes using electrical impedance measurements. At a measurement frequency of 100 kHz non-viable cells, in low-conducting (LC) buffer, show an increase in impedance, due to the resistive effect of the membrane. The opposite effect, an impedance decrease, was observed when a viable cell passed the electrode pair, caused by the presence of the conducting cytoplasm. Moreover, we found that the presence of a viable cell in a droplet also decreased the measured electrical impedance. This impedance change was not visible when a droplet containing a non-viable cell or an empty droplet passed the electrode pair. A non-viable cell in a droplet and an empty droplet were equally classified. Hence, droplets containing (viable) cells can be discriminated from empty droplets. In conclusion, these results provide us with a valuable method to label-free detect and select viable cells in droplets. Furthermore, the proposed method provides the first step towards additional information regarding the encapsulated cells (e.g., size, number, morphology). Moreover, this all-electric approach allows for all-integrated Lab on a Chip (LOC) devices for cell applications using droplet-based platforms.
A Focused Ion Beam (FIB)-patterned silicon mould is used to fabricate elastomeric nanostructures, whose cross-section can be dynamically and reversibly tuned by applying a controlled mechanical stress. Direct-write, based on FIB milling, allows the fabrication of nanostructures with a variety of different geometries, aspect ratio, spacing and distribution offering a higher flexibility compared to other nanopatterning approaches. Moreover, a simple double replication process based on poly(dimethylsiloxane) permits a strong reduction of the fabrication costs that makes this approach well-suited for the production of low cost nanofluidic devices. DNA stretching and single molecule manipulation capabilities of these platforms have been successfully demonstrated.
Nanopore sequencing has the potential to become a direct, fast, and inexpensive DNA sequencing technology. The simplest form of nanopore DNA sequencing utilizes the hypothesis that individual nucleotides of single-stranded DNA passing through a nanopore will uniquely modulate an ionic current flowing through the pore, allowing the record of the current to yield the DNA sequence. We demonstrate that the ionic current through the engineered Mycobacterium smegmatis porin A, MspA, has the ability to distinguish all four DNA nucleotides and resolve single-nucleotides in single-stranded DNA when double-stranded DNA temporarily holds the nucleotides in the pore constriction. Passing DNA with a series of double-stranded sections through MspA provides proof of principle of a simple DNA sequencing method using a nanopore. These findings highlight the importance of MspA in the future of nanopore sequencing.
Microvesicles (MVs) are circular fragments of membrane released from the endosomal compartment as exosomes or shed from the surface membranes of most cell types. An increasing body of evidence indicates that they play a pivotal role in cell-to-cell communication. Indeed, they may directly stimulate target cells by receptor-mediated interactions or may transfer from the cell of origin to various bioactive molecules including membrane receptors, proteins, mRNAs, microRNAs, and organelles. In this review we discuss the pleiotropic biologic effects of MVs that are relevant for communication among cells in physiological and pathological conditions. In particular, we discuss their potential involvement in inflammation, renal disease, and tumor progression, and the evidence supporting a bidirectional exchange of genetic information between stem and injured cells. The transfer of gene products from injured cells may explain stem cell functional and phenotypic changes without the need of transdifferentiation into tissue cells. On the other hand, transfer of gene products from stem cells may reprogram injured cells to repair damaged tissues.
A single-molecule method for sequencing DNA that does not require fluorescent labelling could reduce costs and increase sequencing speeds. An exonuclease enzyme might be used to cleave individual nucleotide molecules from the DNA, and when coupled to an appropriate detection system, these nucleotides could be identified in the correct order. Here, we show that a protein nanopore with a covalently attached adapter molecule can continuously identify unlabelled nucleoside 5'-monophosphate molecules with accuracies averaging 99.8%. Methylated cytosine can also be distinguished from the four standard DNA bases: guanine, adenine, thymine and cytosine. The operating conditions are compatible with the exonuclease, and the kinetic data show that the nucleotides have a high probability of translocation through the nanopore and, therefore, of not being registered twice. This highly accurate tool is suitable for integration into a system for sequencing nucleic acids and for analysing epigenetic modifications.
We show that an electric field can drive single-stranded RNA and DNA molecules through a 2.6-nm diameter ion channel in a lipid bilayer membrane. Because the channel diameter can accommodate only a single strand of RNA or DNA, each polymer traverses the membrane as an extended chain that partially blocks the channel. The passage of each molecule is detected as a transient decrease of ionic current whose duration is proportional to polymer length. Channel blockades can therefore be used to measure polynucleotide length. With further improvements, the method could in principle provide direct, high-speed detection of the sequence of bases in single molecules of DNA or RNA.
Manipulating matter at the nanometre scale is important for many electronic, chemical and biological advances, but present solid-state fabrication methods do not reproducibly achieve dimensional control at the nanometre scale. Here we report a means of fashioning matter at these dimensions that uses low-energy ion beams and reveals surprising atomic transport phenomena that occur in a variety of materials and geometries. The method is implemented in a feedback-controlled sputtering system that provides fine control over ion beam exposure and sample temperature. We call the method "ion-beam sculpting", and apply it to the problem of fabricating a molecular-scale hole, or nanopore, in a thin insulating solid-state membrane. Such pores can serve to localize molecular-scale electrical junctions and switches and function as masks to create other small-scale structures. Nanopores also function as membrane channels in all living systems, where they serve as extremely sensitive electro-mechanical devices that regulate electric potential, ionic flow, and molecular transport across cellular membranes. We show that ion-beam sculpting can be used to fashion an analogous solid-state device: a robust electronic detector consisting of a single nanopore in a Si3N4 membrane, capable of registering single DNA molecules in aqueous solution.
Exosomes shed by tumor cells have been recognized as promising biomarkers for cancer diagnostics due to their unique composition and functions. Quantification of low concentrations of specific exosomes present in very small volumes of clinical samples may be used for non-invasive cancer diagnosis and prognosis. We developed an immunosorbent assay for digital qualification of target exosomes using droplet microfluidics. The exosomes were immobilized on magnetic mircobeads through sandwich ELISA complexes tagged with an enzymatic reporter that produces a fluorescent signal. The constructed beads were further isolated and encapsulated into a sufficient number of droplets to ensure only a single bead was encapsulated in a droplet. Our droplet-based single-exosome-counting enzyme-linked immunoassay (droplet digital ExoELISA) approach enables absolute counting of cancer-specific exosomes to achieve unprecedented accuracy. We were able to achieve a limit of detection (LOD) down to 10 enzyme-labeled exosome complexes per microliter (~10−17 M). We demonstrated the application of the droplet digital ExoELISA platform in quantitative detection of exosomes in plasma samples directly from breast cancer patients. We believe our approach may have the potential for early diagnosis of cancer and accelerate the discovery of cancer exosomal biomarkers for clinical diagnosis.
This paper presents a new method of sensing single molecules and cations by a carbon nanotube (CNT)-based differential resistive pulse sensing (RPS) technique on a nanofluidic chip. A mathematical model for multichannel RPS systems is developed to evaluate the CNT-based RPS signals. Individual cations, rhodamine B dye molecules, and ssDNAs are detected successfully with high resolution and high signal-to-noise ratio. Differentiating ssDNAs with 15 and 30 nucleotides are achieved. The experimental results also show that translocation of negatively charged ssDNAs through a CNT decreases the electrical resistance of the CNT channel, while translocation of positively charged cations and rhodamine B molecules increases the electrical resistance of the CNT. The CNT-based nanofluidic device developed in this work provides a new avenue for single-molecule/ion detection and offers a potential strategy for DNA sequencing.
RPS (Resistive Pulse Sensing) technique is a popular tool for label-free detection of particles. This paper describes a simple, cost-effective PDMS nanofluidic chip for detection and characterization of nanoparticles based on differential RPS technique with high resolution and sensitivity. The chip is composed of two layers of PDMS slabs. Microchannel systems fabricated by photolithography method on the top layer is used for sample loading and differential signal acquisition, and a straight nanochannel on the bottom layer fabricated by an unconventional approach bridging the gap between the microchannels works as an RPS sensing gate. A single-stage differential amplifier is used to amplify the RPS signals when particles or DNAs passing through the sensing gate. It was demonstrated that this nanofluidic RPS chip can detect nanoparticles as small as 23 nm with high SNR (Signal-to-Noise Ratio). The experimental results also show that the device is able to distinguish nanoparticles of smaller size differences such as 60 nm and 83 nm with high resolution, showing superior performance in comparison with the results obtained from a DLS (Dynamic Light Scattering). This differential nano-RPS chip was also applied to detect translocation of dsDNA molecules.
Long DNA molecules can self-entangle into knots. Experimental techniques for observing such DNA knots (primarily gel electrophoresis) are limited to bulk methods and circular molecules below 10 kilobase pairs in length. Here, we show that solid-state nanopores can be used to directly observe individual knots in both linear and circular single DNA molecules of arbitrary length. The DNA knots are observed as short spikes in the nanopore current traces of the traversing DNA molecules and their detection is dependent on a sufficiently high measurement resolution, which can be achieved using high-concentration LiCl buffers. We study the percentage of molecules with knots for DNA molecules of up to 166 kilobase pairs in length and find that the knotting occurrence rises with the length of the DNA molecule, consistent with a constant knotting probability per unit length. Our experimental data compare favourably with previous simulation-based predictions for long polymers. From the translocation time of the knot through the nanopore, we estimate that the majority of the DNA knots are tight, with remarkably small sizes below 100 nm. In the case of linear molecules, we also observe that knots are able to slide out on application of high driving forces (voltage).
The ability to create reproducible and inexpensive nanofluidic chips is essential to the fundamental research and applications of nanofluidics. This paper presents a novel and cost-effective method for fabricating a single nanochannel or multiple nanochannels in PDMS chips with controllable channel size and spacing. Single nanocracks or nanocrack arrays, positioned by artificial defects, are first generated on a polystyrene surface with controllable size and spacing by a solvent-induced method. Two sets of optimal working parameters are developed to replicate the nanocracks onto the polymer layers to form the nanochannel molds. The nanochannel molds are used to make the bi-layer PDMS microchannel-nanochannel chips by simple soft lithography. An alignment system is developed for bonding the nanofluidic chips under an optical microscope. Using this method, high quality PDMS nanofluidic chips with a single nanochannel or multiple nanochannels of sub-100 nm width and height and centimeter length can be obtained with high repeatability.
A long-held goal in sequencing has been to use a voltage-biased nanoscale pore in a membrane to measure the passage of a linear, single-stranded (ss) DNA or RNA molecule through that pore. With the development of enzyme-based methods that ratchet polynucleotides through the nanopore, nucleobase-by-nucleobase, measurements of changes in the current through the pore can now be decoded into a DNA sequence using an algorithm. In this Historical Perspective, we describe the key steps in nanopore strand-sequencing, from its earliest conceptualization more than 25 years ago to its recent commercialization and application.
Enumerating specific cell types from whole blood can be very useful for research and diagnostic purposes-e.g., for counting of CD4 and CD8 T cells in HIV/AIDS diagnostics. We have developed a biosensor based on a differential immunocapture technology to enumerate specific cells in 30 min using 10 μl of blood. This paper provides a comprehensive stepwise protocol to replicate our biosensor for CD4 and CD8 cell counts. The biochip can also be adapted to enumerate other specific cell types such as somatic cells or cells from tissue or liquid biopsies. Capture of other specific cells requires immobilization of their corresponding antibodies within the capture chamber. Therefore, this protocol is useful for research into areas surrounding immunocapture-based biosensor development. The biosensor production requires 24 h, a one-time cell capture optimization takes 6-9 h, and the final cell counting experiment in a laboratory environment requires 30 min to complete.
Coulter counters measure the size of particles in solution by passing them through an orifice and measuring a resistive pulse, i.e., a drop in the ionic current flowing between two electrodes placed either side of the orifice. The magnitude of the pulse gives information on the size of the particle; however, accuracy is limited by variability in the path of the translocation, due to the Brownian motion of the particle. We present a simple-yet-powerful modified Coulter counter that uses programmable data acquisition hardware to switch the voltage after sensing the resistive pulse of a nanoparticle passing through the orifice of a nanopipette. Switching the voltage reverses the direction of the driving force on the particle, and when this detect-switch cycle is repeated, allows us to pass an individual nanoparticle through the orifice thousands of times. By measuring individual particles more than one hundred times per second we rapidly determine the distribution of the resistive pulses for each particle, which allows us to accurately determine mean pulse amplitude and deliver considerably improved size resolution over a conventional Coulter counter. We show that single polystyrene nanoparticles can be shuttled back and forth and monitored for minutes, leading to a precisely determined mean blocking current equating to sub-Angstrom size resolution.
Nanopores in graphene
membranes can potentially offer unprecedented spatial resolution for single molecule sensing, but their fabrication has thus far been difficult, poorly scalable, and prone to contamination. We demonstrate an in-situ
fabrication method that nucleates and controllably enlarges nanopores in electrolyte solution by applying ultra-short, high-voltage pulses across the graphene
membrane. This method can be used to rapidly produce graphene
nanopores with subnanometer size accuracy in an apparatus free of nanoscale beams or tips.
Electrophoretic mobilities and particle sizes of individual Hepatitis B Virus (HBV) capsids were measured in nanofluidic channels with two nanopores in series. The channels and pores had three-dimensional topography and were milled directly in glass substrates with a focused ion beam instrument assisted by an electron flood gun. The nanochannel between the two pores was 300 nm wide, 100 nm deep, and 2.5 μm long, and the nanopores at each end had dimensions 45 nm wide, 45 nm deep, and 400 nm long. With resistive-pulse sensing, the nanopores fully resolved pulse amplitude distributions of T = 3 HBV capsids (32 nm outer diameter) and T = 4 HBV capsids (35 nm outer diameter) and had sufficient peak capacity to discriminate intermediate species from the T = 3 and T = 4 capsid distributions in an assembly reaction. Because the T = 3 and T = 4 capsids have a wiffle-ball geometry with a hollow core, the observed change in current due to the capsid transiting the nanopore is proportional to the volume of electrolyte displaced by the volume of capsid protein, not the volume of the entire capsid. Both the signal-to-noise ratio of the pulse amplitude and resolution between the T = 3 and T = 4 distributions of the pulse amplitudes increase as the electric field strength is increased. At low field strengths, transport of the larger T = 4 capsid through the nanopores is hindered relative to the smaller T = 3 capsid due to interaction with the pores, but at sufficiently high field strengths, the T = 3 and T = 4 capsids had the same electrophoretic mobilities (7.4 × 10(-5) cm(2) V(-1) s(-1)) in the nanopores and in the nanochannel with the larger cross-sectional area.
We study translocations of gold nanoparticles and nanorods through silicon nitride nanopores and present a method for determining the surface charge of nanorods from the magnitude of the ionic current change as nanorods pass through the pore. Positively-charged nanorods and spherical nanoparticles with average diameters 10 nm and average nanorod lengths between 44 and 65 nm were translocated through 40 nm thick nanopores with diameters between 19 and 27 nm in 1, 10, or 100 mM KCl solutions. Nanorod passage through the nanopores decreases ion current in larger diameter pores, as in the case of typical Coulter counters, but it increases ion current in smaller diameter nanopores, likely because of the interaction of the nanopore's and nanoparticle's double layers. The presented method predicts a surface charge of 26 mC/m(2) for 44 nm long gold nanorods and 18 mC/m(2) for 65 nm long gold nanorods and facilitates future studies of ligand coverage and surface charge effects in anisotropic particles.
This paper describes a fundamental study of the effect of electrostatic interactions on the resistive pulse waveshape associated with translocation of charged nanoparticles through a conical-shaped, charged glass nanopore. In contrast to single-peak resistive pulses normally associated with resistive-pulse methods, biphasic pulses, in which the normal current decrease is preceded by a current increase, were observed in the current–time recordings when a high negative potential (lower than −0.4 V) is applied between the pore interior and the external solution. The biphasic pulse is a consequence of the offsetting effects of an increased ion conductivity induced by the surface charge of the translocating particle and the current decrease due to the volume exclusion of electrolyte solution by the particle. Finite-element simulations based on the coupled Poisson–Nernst–Planck equations and a particle trajectory calculation successfully capture the evolution of the waveshape from a single resistive pulse to a biphasic response as the applied voltage is varied. The simulation results demonstrate that the surface charges of the nanopore and the particle are responsible for the voltage-dependent shape evolution. Additionally, the use of high ionic strength solution or high pressures to drive particle translocation was found to eliminate the biphasic response. The former is due to the screening of the electrical double layer, while the latter results from the solution flow preventing formation of an equilibrium double layer ion distribution within the nanopore, similar to the previously reported elimination of ion current rectification when solution flows through a nanopore.
The feasibility of using quartz nanopipettes as simple and cost-effective Coulter counters for calibration-less quantitation and sizing of nanoparticles by resistive pulsing sensing (RPS) was investigated. A refined theory was implemented to calculate the size distribution of nanoparticles based on the amplitude of resistive pulses caused by their translocation through nanopipettes of known geometry. The RPS provided diameters of monodisperse latex nanoparticles agreed within the experimental error with those measured by using scanning electron microscopy (SEM), dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). The nanopipette-based counter, by detecting individual nanoparticles could resolve with similar resolution as SEM mixtures of monodisperse nanoparticles having partially overlapping size distributions, which could not be discriminated by DLS or NTA. Furthermore, by calculating the hydrodynamic resistance of the nanopipettes and consequently the volume flow through the tip enabled for the first time the calibration-less determination of nanoparticle concentrations with nanopipettes. The calibration-less methodology is applied to sizing and quantitation of inactivated poliovirus of ca. 26 nm diameter, which is the smallest size spherical shape virus ever measured by resistive pulse sensing.
Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer-associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2 ) or normoxic (21% O2 ) conditions, and exosomes secreted under hypoxic (Exo(Hypoxic) ) and normoxic (Exo(Normoxic) ) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that Exo(Hypoxic) have smaller average size as compared to Exo(Normoxic) . Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70), and Annexin II in Exo(Hypoxic) compared to Exo(Normoxic) . Co-culturing with Exo(Hypoxic) increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. Exo(Hypoxic) also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α-SMA (a CAF biomarker) expression in PrSC. Compared to Exo(Normoxic) , Exo(Hypoxic) showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF-β2, TNF1α, IL6, TSG101, Akt, ILK1, and β-catenin). Furthermore, proteome analysis revealed a higher number of proteins in Exo(Hypoxic) (160 proteins) compared to Exo(Normoxic) (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, Exo(Hypoxic) targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that Exo(Hypoxic) are loaded with unique proteins that could enhance invasiveness, stemness, and induce microenvironment changes; thereby, promoting PCA aggressiveness.
This paper reports a method of detecting nanoparticles using a microfluidic differential Resistive Pulse Sensor (RPS). Using a single microfluidic channel with two detecting arm channels placed at the two ends of the sensing section, the microfluidic differential RPS can achieve a high sensitivity allowing the detection of nanometer size particles. Two-stage differential amplification is used to further increase the signal-to-noise ratio. This method is able to detect nanoparticles of 490nm and 220nm on a microfluidic chip. The method described in this paper is simple and can be applied to develop a compact device without the need of bulky, sophisticated electronic instruments or complicated nano-fabrication processes.
It has recently been recognized that solid-state nanopores in single-atomic-layer graphene membranes can be used to electronically detect and characterize single long charged polymer molecules. We have now fabricated nanopores in single-layer graphene that are closely matched to the diameter of a double-stranded DNA molecule. Ionic current signals during electrophoretically driven translocation of DNA through these nanopores were experimentally explored and theoretically modeled. Our experiments show that these nanopores have unusually high sensitivity (0.65 nA/Å) to extremely small changes in the translocating molecule's outer diameter. Such atomically short graphene nanopores can also resolve nanoscale-spaced molecular structures along the length of a polymer, but do so with greatest sensitivity only when the pore and molecule diameters are closely matched. Modeling confirms that our most closely matched pores have an inherent resolution of ≤0.6 nm along the length of the molecule.
The first electronic measurement of DNA translocation through ultrathin BN nanopores is demonstrated. BN nanopores show much higher detection sensitivity compared with SiN nanopores. BN has as high a spatial resolution as graphene. The ultrathin BN nanopores provide substantial opportunities in realizing high-spatial-sensitivity nanopore electrical devices for various applications.
Ion current through a single-walled carbon nanotube (SWCNT) was monitored at the same time as fluorescence was recorded from charged dye molecules translocating through the SWCNT. Fluorescence bursts generally follow ion-current peaks with a delay time consistent with diffusion from the end of the SWCNT to the fluorescence collection point. The fluorescence amplitude distribution of the bursts is consistent with single molecule signals. Thus each peak in the ion current flowing through the SWCNT is associated with the translocation of a single molecule. Ion current peaks (as opposed to blockades) were produced by both positively (Rhodamine 6G) and negatively (Alexa 546) charged molecules, showing that the charge filtering responsible for the current bursts is caused by the molecules themselves.
This paper reports a lab-on-a-chip device that can automatically detect and sort particles based on their size differences with a high resolution. The PDMS-glass microfluidic chip is made by soft lithography technique. A differential resistive pulse sensor (RPS) is employed to electrically detect the sizes of the particles in electroosmotic flow generated by applying DC voltages across channels. The detected PRS signals, whose amplitudes are proportional to particles' sizes, will automatically trigger the sorting process that is controlled by applying a voltage pulse (36V) whenever a target particle is detected. This method was applied to automatically detect and sort polystyrene particles and micro algae in aqueous solutions. Sorting 5μm polymer particle from a mixture of 4μm and 5μm polystyrene particles in aqueous solution, i.e., 1μm sorting resolution, was demonstrated. The device described in this paper is simple, automatic and label-free with high sorting resolution. It has wide applications in sample pretreatment and target particles detection.
We have used micromolding techniques to embed a nanoscale pore in PDMS. This novel design allows rapid and reproducible fabrication of pores, is extremely flexible, and can be modified both structurally and chemically for a variety of single-molecule detection applications. We demonstrate the capabilities of the device to sense electronically single DNA molecules. Because ion-channel proteins are so finely tuned to respond to a single molecule, they have served as a model for developing nanopore devices for biomolecular sensing. 1 Two strategies for engineered nanoporesstransmembrane protein pores suspended in lipid bilayers 2-5 and molecular-scaled holes in silicon nitride 6 shave achieved success in detecting single biological molecules; however, further development of these strategies is impeded by several technological barriers, including difficulties in creating an effective pore or array of pores and stabilizing it over a period of time. Here we report on a fundamentally different artificial nanopore that can be fabricated with great ease and control using micromolding techniques, is capable of sensing single molecules of unlabeled λ-phage DNA, and provides op-portunities for diverse single-molecule detection applications. Figure 1 shows one of our devices: a pore of length 3 µm and diameter 200 nm connecting two 5-µm-deep reservoirs. Well-established lithographic techniques are used to create a negative master of the pore and reservoirs, which is subsequently cast into a poly(dimethylsiloxane) (PDMS) slab. The master is created in two steps: first, electron-beam lithography is used to pattern a 200-nm-wide, 200-nm-thick polystyrene line on a silicon substrate, creating the negative of the pore. Next, photolithography is used to pattern an SU-8 photoresist on the substrate to form the negatives of the reservoirs. Both resists (polystyrene and SU-8) are excep-tionally durable once cross linked, allowing us to reuse each master indefinitely. Following standard micromolding tech-niques, 7 we pour PDMS (Sylgard 184) over the master and cure it at 80 °C for at least 24 h. The PDMS slab is then removed from the master and sealed to a glass substrate that has previously defined platinum electrodes. The device is now complete and can be wet with the solution to be studied. Molecules in the solution are driven through the pore either electrophoretically or by applying pressure to one of the reservoirs. When in the pore, the molecules partially block the flow of current, leading to transient increases in the pore's electrical resistance. Molecular sensing is accomplished by performing a four-point measurement of the electrical current through the pore using the platinum electrodes. The current is low-pass filtered below 0.3 ms in rise time and is sampled at 1 kHz using a voltmeter. To demonstrate the sensing capabilities of our nanopore, we have measured solutions of 2.5 µg/mL λ-phage DNA in a 0.1 M KCl, 2 mM Tris (pH 8.4) buffer. Typical traces of measured current are shown in Figure 2. The striking downward peaks, of height 10-30 pA and width 2-10 ms, correspond to individual molecules of DNA passing through the pore. In contrast, such peaks are absent when measuring only buffer. We further note that peaks are present only when using pores with diameters of 300 nm or less.
A method for detecting and counting pollen particles based on Coulter counting principle is presented. This approach also provides information on the size and surface charges of the micro particles, allowing for preliminary differentiation of pollens from other micro particles. Three samples are studied: polymethyl methacrylate particles, tree pollens from Juniperus Scopulorum and grass pollens from Secale Cerale. The samples, suspended in diluted KCl aqueous solutions in an electrochemical cell, were allowed to pass through a microchannel and the conductance of the microchannel was sampled with a Gamry® Potentiostat. The changes in the conductance due to the passing of the micro particles was thus recorded and analyzed. The experimental results showed that tree pollens and grass pollens display distinctive behaviors. The phenomena may be attributed to the differences in the surface characteristics of the pollens and is potentially useful for counting and differentiating different micro particles.
We report fabrication and characterization of nanochannel devices with two nanopores in series for resistive-pulse sensing of hepatitis B virus (HBV) capsids. The nanochannel and two pores are patterned by electron beam lithography between two microchannels and etched by reactive ion etching. The two nanopores are 50-nm wide, 50-nm deep, and 40-nm long and are spaced 2.0-μm apart. The nanochannel that brackets the two pores is 20× wider (1 μm) to reduce the electrical resistance adjacent to the two pores and to ensure the current returns to its baseline value between resistive-pulse events. Average pulse amplitudes differ by <2% between the two pores and demonstrate that the fabrication technique is able to produce pores with nearly identical geometries. Because the two nanopores in series sense single particles at two discrete locations, particle properties, e.g., electrophoretic mobility, are determined from the pore-to-pore transit time.
We report characterization of hepatitis B virus (HBV) capsids by resistive-pulse sensing through single track-etched conical nanopores formed in poly(ethylene terephthalate) membranes. The pores were ∼40 nm in diameter at the tip, and the pore surface was covalently modified with triethylene glycol to reduce surface charge density, minimize adsorption of the virus capsids, and suppress electroosmotic flow in the pore. The HBV capsids were assembled in vitro from Cp149, the assembly domain of HBV capsid protein. Assembled T = 3 (90 Cp149 dimer) and T = 4 (120 dimer) capsids are 31 and 36 nm in diameter, respectively, and were easily discriminated by monitoring the change in current as capsids passed through an electrically biased pore. The ratio of the number of T = 3 to T = 4 capsids transiting a pore did not reflect actual concentrations, but favored transport of smaller T = 3 capsids. These results combined with longer transit times for the T = 4 capsids indicated that the capsids must overcome an entropic barrier to enter a pore.
We investigate the translocation of lambda-DNA molecules through resistive-pulse polydimethylsiloxane (PDMS) nanopore sensors. Single molecules of lambda-DNA were detected as a transient current increase due to the effect of DNA charge on ionic current through the pore. DNA translocation was found to deviate from a Poisson process when the interval between translocations was comparable to the duration of translocation events, suggesting that translocation was impeded during the presence of another translocating molecule in the nanopore. Characterization of translocation at different voltage biases revealed that a critical voltage was necessary to drive DNA molecules through the nanopore. Above this critical voltage, frequency of translocation events was directly proportional to DNA concentration and voltage bias, suggesting that transport of DNA from the solution to the nanopore was the rate limiting step. These observations are consistent with experimental results on transport of DNA through nanopores and nanoslits and the theory of hydrodynamically driven polymer flow in pores.