Chiral Graphene Quantum Dots Enhanced Drug Loading into Small Extracellular Vesicles.

As nanoscale extracellular vesicles secreted by cells, small extracellular vesicles (sEVs) have enormous potential as safe and effective vehicles to deliver drugs into lesion locations. Despite promising advances with sEV-based drug delivery systems, there are still challenges to drug loading into sEVs, which hinder the clinical applications of sEVs. Herein, we report an exogenous drug-agnostic chiral graphene quantum dots (GQDs) sEV-loading platform, based on chirality matching with the sEV lipid bilayer. Both hydrophobic and hydrophilic chemical and biological drugs can be functionalized or adsorbed onto GQDs by π-π stacking and van der Waals interactions. By tuning the ligands and GQD size to optimize its chirality, we demonstrate drug loading efficiency of 66.3% and 64.1% for doxorubicin and siRNA, which is significantly higher than other reported sEV loading techniques.

Quantifying PON1 on HDL with nanoparticle-gated electrokinetic membrane sensor for accurate cardiovascular risk assessment.

Cardiovascular disease-related deaths (one-third of global deaths) can be reduced with a simple screening test for better biomarkers than the current lipid and lipoprotein profiles. We propose using a highly atheroprotective subset of HDL with colocalized PON1 (PON1-HDL) for superior cardiovascular risk assessment. However, direct quantification of HDL proteomic subclasses are complicated by the peroxides/antioxidants associated with HDL interfering with redox reactions in enzymatic calorimetric and electrochemical immunoassays. Hence, we developed an enzyme-free Nanoparticle-Gated Electrokinetic Membrane Sensor (NGEMS) platform for quantification of PON1-HDL in plasma within 60 min, with a sub-picomolar limit of detection, 3-4 log dynamic range and without needing sample pretreatment or individual-sample calibration. Using NGEMS, we report our study on human plasma PON1-HDL as a cardiovascular risk marker with AUC~0.99 significantly outperforming others (AUC~0.6-0.8), including cholesterol/triglycerides tests. Validation for a larger cohort can establish PON1-HDL as a biomarker that can potentially reshape cardiovascular landscape.

Chiral Graphene Quantum Dots Enhanced Drug Loading into Exosomes.

As nanoscale extracellular vesicles secreted by cells, exosomes have enormous potential as safe and effective vehicles to deliver drugs into lesion locations. Despite promising advances with exosome-based drug delivery systems, there are still challenges to drug loading into exosome, which hinder the clinical applications of exosomes. Herein, we report an exogenous drug-agnostic chiral graphene quantum dots (GQDs) exosome-loading platform, based on chirality matching with the exosome lipid bilayer. Both hydrophobic and hydrophilic chemical and biological drugs can be functionalized or adsorbed onto GQDs by π-π stacking and van der Waals interactions. By tuning the ligands and GQD size to optimize its chirality, we demonstrate drug loading efficiency of 66.3% and 64.1% for Doxorubicin and siRNA, which is significantly higher than other reported exosome loading techniques.

Electrodeposited magnetic nanoporous membrane for high-yield and high-throughput immunocapture of extracellular vesicles and lipoproteins.

Superparamagnetic nanobeads offer several advantages over microbeads for immunocapture of nanocarriers (extracellular vesicles, lipoproteins, and viruses) in a bioassay: high-yield capture, reduction in incubation time, and higher capture capacity. However, nanobeads are difficult to "pull-down" because their superparamagnetic feature requires high nanoscale magnetic field gradients. Here, an electrodeposited track-etched membrane is shown to produce a unique superparamagnetic nano-edge ring with multiple edges around nanopores. With a uniform external magnetic field, the induced monopole and dipole of this nano edge junction combine to produce a 10× higher nanobead trapping force. A dense nanobead suspension can be filtered through the magnetic nanoporous membrane (MNM) at high throughput with a 99% bead capture rate. The yield of specific nanocarriers in heterogeneous media by nanobeads/MNM exceeds 80%. Reproducibility, low loss, and concentration-independent capture rates are also demonstrated. This MNM material hence expands the application of nanobead immunocapture to physiological samples.

Correction: Ultrasensitive fluorescent aptasensor for CRP detection based on the RNase H assisted DNA recycling signal amplification strategy.

[This corrects the article DOI: 10.1039/C9RA01352K.].

Correction: A fluorescent biosensor for cardiac biomarker myoglobin detection based on carbon dots and deoxyribonuclease I-aided target recycling signal amplification.

[This corrects the article DOI: 10.1039/C8RA09459D.].

Development of a Multi-target Protein Biomarker Assay for Circulating Tumor Cells.

We report a highly sensitive and selective CNT-switch liquid biopsy platform that detects and quantifies protein biomarker expressions from circulating tumor cells in blood for early detection of metastatic breast cancer and its relapse. This platform first isolates and enriches more than 99% of tumor cells with an off-chip micro-size membrane filtration technique and then conducts on-chip detection of the membrane and internal protein biomarkers of the tumor cells with high sensitivity and selectivity. High sensitivity is achieved with complete association of the antibody-antigen-antibody (Ab-Ag-Ab) complex by precisely and rapidly assembling carbon nanotubes (CNTs) across two parallel electrodes via sequential DC electrophoresis and dielectrophoresis (DEP) deposition. Each bridged CNT acts as a switch that connects the electrodes and closes the circuit to generate an electrical signal. The high selectivity is achieved with a critical hydrodynamic shear rate that irreversibly removes non-target linkers of the aligned CNTs. At present, we are able to detect the protein biomarkers from 5 spiked breast cancer tumor cells of different types within 7.5 ml of human blood samples. This demonstrates the potential of this platform as an inexpensive and noninvasive alternative to MRI scans and tissue biopsies currently used to detect early metastatic breast cancer and its relapse.

Nanoparticle-assisted detection of nucleic acids in a polymeric nanopore with a large pore size.

Rapid and accurate detection of nucleic acids is of paramount importance in many fields, including medical diagnosis, gene therapy and virus identification. In this work, by taking advantage of two DNA hybridization probes, one of which was immobilized on the surface of gold nanoparticles, while the other was free in solution, detection of short length nucleic acids was successfully achieved using a large size (20 nm tip diameter) polyethylene terephythalate (PET) nanopore. The sensor was sensitive and selective: DNA samples with concentrations at as low as 0.5 nM could be detected within minutes and the number of mismatches can be discerned from the translocation frequency. Furthermore, the nanopore can be repeatedly used many times. Our developed large-size nanopore sensing platform offers the potential for fieldable/point-of-care diagnostic applications.

Elliptical Pipette Generated Large Microdroplets for POC Visual ddPCR Quantification of Low Viral Load.

Rapid point-of-care (POC) quantification of low virus RNA load would significantly reduce the turn-around time for the PCR test and help contain a fast-spreading epidemic. Herein, we report a droplet digital PCR (ddPCR) platform that can achieve this sensitivity and rapidity without bulky lab-bound equipment. The key technology is a flattened pipette tip with an elliptical cross-section, which extends a high aspect-ratio microfluidic chip design to pipette scale, for rapid (<5 min) generation of several thousand monodispersed droplets ∼150 to 350 µm in size with a CV of ∼2.3%. A block copolymer surfactant (polyoxyalkylene F127) is used to stabilize these large droplets in oil during thermal cycling. At this droplet size and number, positive droplets can be counted by eye or imaged by a smartphone with appropriate illumination/filtering to accurately quantify up to 100 target copies. We demonstrate with 2019 nCoV-PCR assay LODs of 3.8 copies per 20 µL of sample and a dynamic range of 4-100 copies. The ddPCR platform is shown to be inhibitor resistant with spiked saliva samples, suggesting RNA extraction may not be necessary. It represents a rapid 1.5-h POC quantitative PCR test that requires just a pipette equipped with elliptical pipette tip, a commercial portable thermal cycler, a smartphone, and a portable trans-illuminator, without bulky and expensive micropumps and optical detectors that prevent POC application.

Slowing down DNA translocation through solid-state nanopores by edge-field leakage.

Solid-state nanopores allow high-throughput single-molecule detection but identifying and even registering all translocating small molecules remain key challenges due to their high translocation speeds. We show here the same electric field that drives the molecules into the pore can be redirected to selectively pin and delay their transport. A thin high-permittivity dielectric coating on bullet-shaped polymer nanopores permits electric field leakage at the pore tip to produce a voltage-dependent surface field on the entry side that can reversibly edge-pin molecules. This mechanism renders molecular entry an activated process with sensitive exponential dependence on the bias voltage and molecular rigidity. This sensitivity allows us to selectively prolong the translocation time of short single-stranded DNA molecules by up to 5 orders of magnitude, to as long as minutes, allowing discrimination against their double-stranded duplexes with 97% confidence.

Chemically functionalized conical PET nanopore for protein detection at the single-molecule level.

Proteins are essential for all living organisms, and perform a wide variety of functions in the cell and human body, including structural, mechanical, biochemical, and signaling. Since proteins can serve as valuable biomarkers for health status and diseases states, and enable personalized medicine, sensitive and rapid detection of proteins is of paramount importance. Herein, we report a chemically functionalized conical shaped poly-(ethylene terephthalate) nanopore (PET nanopore) as a stochastic sensing element for detection of proteins at the single-molecule level. We demonstrate that the PET nanopore sensor is not only sensitive and selective, but also can differentiate proteins rapidly, offering the potential for label-free protein detection and characterization. Our developed PET nanopore sensing strategy not only provides a general platform for exploring fundamental protein dynamics and rapid detection of proteins at the single-molecule level, but also opens new avenues toward advanced deeper understanding of enzymes, development of more efficient biosensing technologies, and constructing novel biomimetic nanopore systems.

Resistive amplitude fingerprints during translocation of linear molecules through charged solid-state nanopores.

We report the first analytical theory on the amplitude of resistive signals during molecular translocation through charged solid-state nanopores with variable cross-sectional area and piecewise-constant surface charge densities. By providing closed-form explicit algebraic expressions for the concentration profiles inside charged nanopores, this theory allows the prediction of baseline and translocation resistive signals without the need for numerical simulation of the electrokinetic phenomena. A transversely homogenized theory and an asymptotic expansion for weakly charged pores capture DC or quasi-static rectification due to field-induced intrapore concentration polarization (as a result of pore charge inhomogeneity or a translocating molecule). This theory, validated by simulations and experiments, is then used to explain why the amplitude of a single stranded DNA molecule can be twice as high as the amplitude of its double stranded counterpart. It also suggests designs for intrapore concentration polarization and volume exclusion effects that can produce biphasic and other amplitude fingerprints for high-throughput and yet discriminating molecular identification.

Liquid biopsy technologies based on membrane microfluidics: High-yield purification and selective quantification of biomarkers in nanocarriers.

Liquid biopsy, screening cancer non-invasively and frequently by detecting and quantifying molecular markers in physiological fluids, would significantly improve cancer survival rate but it remains a distant goal. The key obstacles presented by the highly heterogeneous samples are rapid/high-yield purification and precise/selective marker capture by their antibody and oligo probes. As irregular expressions of these molecular biomarkers are the key signals, quantifying only those from the cancer cells would greatly enhance the performance of the screening tests. The recent discovery that the biomarkers are carried by nanocarriers, such as exosomes, with cell-specific membrane proteins suggests that such selection may be possible, although a new suite of fractionation and quantification technologies would need to be developed. Although under-appreciated, membrane microfluidics has made considerable contributions to resolving these issues. We review the progress made so far, based on ion-selective, track-etched, and gel membranes and advanced electrophoretic and nano-filtration designs, in this perspective and suggest future directions.

An electrochemical aptasensing platform for carbohydrate antigen 125 based on the use of flower-like gold nanostructures and target-triggered strand displacement amplification.

An electrochemical aptasensing method is described for the determination of the biomarker CA125. It combines aptamer recognition and target-triggered strand displacement amplification. Flower like gold nanostructures were electrodeposited on a screen-printed carbon electrode to increase the sensor surface, to assemble more toehold-containing hairpin probe 1 (Hp1), and to improve the accessibility for DNA strands. Under the optimal conditions, this assay has a linear response in the 0.05 to 50 ng•mL-1 CA125 concentration range, with a low detection limit of 5.0 pg•mL-1. This method is specific and stable. It was successfully applied to the detection of CA125 in spiked biological samples, with recoveries between 82.5% and 104.1%. Graphical abstract.

Ultrasensitive fluorescent aptasensor for CRP detection based on the RNase H assisted DNA recycling signal amplification strategy.

An aptamer-based method for the ultrasensitive fluorescence detection of C-reactive protein (CRP) was developed using the ribonuclease H (RNase H) assisted DNA recycling signal amplification strategy. In this assay, CRP can specifically bind to the aptamer of CRP and the DNA chain of P1 is released from the aptamer/P1 (Ap/P1) complexes. After the addition of the fluorescence labeled (5-FAM) RNA, P1 hybridizes with fluorescence labeled RNA to form a P1/RNA double strand. When RNase H is added, the RNA with fluorescence labeling in the double strand is specifically cut into nucleotide fragments, which cannot be adsorbed on the surface of the GO, so as to generate a fluorescence signal. In the absence of CRP, fluorescence labeled RNA cannot hybridize with P1 to form double strands, which is able to directly adsorb on the surface of GO, resulting in no fluorescence signal. The detection limit is as low as 0.01 ng mL-1, with a linear dynamic range from 50 pg mL-1 to 100 ng mL-1. This sensor is able to detect CRP in spiked human serum, urine and saliva. Thus, it shows a great application prospect in disease diagnosis and prognosis.

A fluorescent biosensor for cardiac biomarker myoglobin detection based on carbon dots and deoxyribonuclease I-aided target recycling signal amplification.

A sensitive biosensor using carbon dots and deoxyribonuclease I-aided target recycling signal amplification has been developed to detect myoglobin (MB), which is an important cardiac biomarker and plays a major role in the diagnosis of acute myocardial infarction (AMI). Here, in the absence of MB, the MB aptamer (Ap) is absorbed on the surface of carbon dots (CDs) through π-π stacking interactions, resulting in quenching of the fluorescent label by forming CD-aptamer complexes. Upon adding MB, the Ap sequences could be specifically recognized by MB, leading to the recovery of quenched fluorescence. Thus, quantitative evaluation of MB concentration has been achieved in a broad range from 50 pg mL-1 to 100 ng mL-1, and the detection limit is as low as 20 pg mL-1. This strategy is capable of specific and sensitive detection of MB in human serum, urine, and saliva and can be used for the diagnosis of AMI in the future.

A shear-enhanced CNT-assembly nanosensor platform for ultra-sensitive and selective protein detection.

Detection and quantification of low-concentration proteins in heterogeneous media are generally plagued by two distinct obstacles: lack of sensitivity due to high dissociation equilibrium constant KD and non-specificity due to an abundance of non-targets with similar KD. Herein, we report a nanoscale protein-sensing platform with a non-equilibrium on-off switch that employs dielectrophoretic and hydrodynamic shear forces to overcome these thermodynamic limitations with irreversible kinetics. The detection sensitivity is achieved with complete association of the antibody-antigen-antibody (Ab-Ag-Ab) complex by precisely and rapidly assembling carbon nanotubes (CNT) across two parallel electrodes via sequential DC electrophoresis and AC dielectrophoresis (DEP), and with single-CNT electron tunneling conductance. The high selectivity is achieved with a critical hydrodynamic shear rate between the activated dissociation shear rates of target and non-target linkers of the aligned CNTs. We are able to reach detection limits of 100 attomolar (aM) and 10 femtomolar (fM) in pure samples for two ELISA assays with low and high dissociation constant: biotin/streptavidin (10 fM) and HER2/HER2 antibody (0.44 ± 0.07nM), respectively. For both models, irreversible capture and shearing allow us to tune the dynamic range up to 5 decades by increasing the CNT numbers. We also demonstrate in spiked serum sample high selectivity towards target HER2 proteins against non-target HER2 isoform of a similar KD. The detection limit for HER2 in serum is lower than 100fM.

Universal Scaling of Robust Thermal Hot Spot and Ionic Current Enhancement by Focused Ohmic Heating in a Conic Nanopore.

A stable nanoscale thermal hot spot, with temperature approaching 100 °C, is shown to be sustained by localized Ohmic heating of a focused electric field at the tip of a slender conic nanopore. The self-similar (length-independent) conic geometry allows us to match the singular heat source at the tip to the singular radial heat loss from the slender cone to obtain a self-similar steady temperature profile along the cone and the resulting ionic current conductance enhancement due to viscosity reduction. The universal scaling, which depends only on a single dimensionless parameter Z, collapses the measured conductance data and computed temperature profiles in ion-track conic nanopores and conic nanopipettes. The collapsed numerical data reveal universal values for the hot-spot location and temperature in an aqueous electrolyte.

Future microfluidic and nanofluidic modular platforms for nucleic acid liquid biopsy in precision medicine.

Nucleic acid biomarkers have enormous potential in non-invasive diagnostics and disease management. In medical research and in the near future in the clinics, there is a great demand for accurate miRNA, mRNA, and ctDNA identification and profiling. They may lead to screening of early stage cancer that is not detectable by tissue biopsy or imaging. Moreover, because their cost is low and they are non-invasive, they can become a regular screening test during annual checkups or allow a dynamic treatment program that adjusts its drug and dosage frequently. We briefly review a few existing viral and endogenous RNA assays that have been approved by the Federal Drug Administration. These tests are based on the main nucleic acid detection technologies, namely, quantitative reverse transcription polymerase chain reaction (PCR), microarrays, and next-generation sequencing. Several of the challenges that these three technologies still face regarding the quantitative measurement of a panel of nucleic acids are outlined. Finally, we review a cluster of microfluidic technologies from our group with potential for point-of-care nucleic acid quantification without nucleic acid amplification, designed to overcome specific limitations of current technologies. We suggest that integration of these technologies in a modular design can offer a low-cost, robust, and yet sensitive/selective platform for a variety of precision medicine applications.

A capacitive-pulse model for nanoparticle sensing by single conical nanochannels.

Nanochannel based devices have been widely used for single-molecule detection. The detection usually relies on the resistive-pulse model, where the change of the monitored current depends on the physical volumetric blocking of the nanochannel by the analyte. However, this mechanism requires that the nanochannel diameter should not be much larger than the analyte size, because, otherwise, the resultant current change would be too small to detect, and therefore poses particular challenges for the fabrication of nanochannels. To circumvent this issue, in this report, we propose a different mechanism of capacitive-pulse model, where the transport signals can be significantly magnified by the capacitive effect of the nanochannel. We experimentally demonstrate that current pulses with an averaged peak height of 0.87 nA can be achieved for transporting 60 nm nanoparticles through a conical nanochannel device, whereas the traditional resistive-pulse model only predicts one-order-of-magnitude lowered value. With further comprehensive simulation, the dependence of this effect on the nanochannel geometry as well as the surface charge density for both the nanochannel and the analyte is predicted, which would provide important guidance for better designing of the nanochannel-based sensors.