On-chip microscale isoelectric focusing enhances protein detection limit.

Enhancing the detection limit in protein analysis is essential for a wide range of biomedical applications. In typical fluorescent protein assays, this limit is constrained by the detection capacity of the photon detector. Here, we develop an approach that significantly enhances the protein detection threshold by using microscale isoelectric focusing implemented directly at the detection site on a protein sensor chip. We demonstrate that by electrically generating a localized pH environment within a radius of ∼60 µm, protein molecules can be concentrated within this range and be detected at levels over four times lower than those achieved by measurements without on-chip isoelectric focusing. We find that this detection-limit enhancement results from a dual effect: the concentrating of the protein molecules and a reduction in the diffusion-induced fluctuation. Our approach offers a simple, yet highly effective ultra-low-power all-electronic solution for substantially improving protein analysis detection limits for diverse applications, including healthcare, clinical diagnostics, and therapeutics.

Nanomechanoelectrical approach to highly sensitive and specific label-free DNA detection.

Electronic detection of DNA oligomers offers the promise of rapid, miniaturized DNA analysis across various biotechnological applications. However, known all-electrical methods, which solely rely on measuring electrical signals in transducers during probe-target DNA hybridization, are prone to nonspecific electrostatic and electrochemical interactions, subsequently limiting their specificity and detection limit. Here, we demonstrate a nanomechanoelectrical approach that delivers ultra-robust specificity and a 100-fold improvement in detection limit. We drive nanostructural DNA strands tethered to a graphene transistor to oscillate in an alternating electric field and show that the transistor-current spectra are characteristic and indicative of DNA hybridization. We find that the inherent difference in pliability between unpaired and paired DNA strands leads to the spectral characteristics with minimal influence from nonspecific electrostatic and electrochemical interactions, resulting in high selectivity and sensitivity. Our results highlight the potential of high-performance DNA analysis based on miniaturized all-electronic settings.

Defect Healing in Graphene via Rapid Thermal Annealing with Polymeric "Nanobandage".

Overcoming throughput challenges in current graphene defect healing processes, such as conventional thermal annealing, is crucial for realizing post-silicon device fabrication. Herein, a new time- and energy-efficient method for defect healing in graphene is reported, utilizing polymer-assisted rapid thermal annealing (RTA). In this method, a nitrogen-rich, polymeric "nanobandage" is coated directly onto graphene and processed via RTA at 800 °C for 15 s. During this process, the polymer matrix is cleanly degraded, while nitrogen released from the nanobandage can diffuse into graphene, forming nitrogen-doped healed graphene. To study the influence of pre-existing defects on graphene healing, lattice defects are purposefully introduced via electron beam irradiation and investigated by Raman microscopy. X-ray photoelectron spectroscopy reveals successful healing of graphene, observing a maximum doping level of 3 atomic nitrogen % in nanobandage-treated samples from a baseline of 0-1 atomic % in non-nanobandage treated samples. Electrical transport measurements further indicate that the nanobandage treatment recovers the conductivity of scanning electron microscope-treated defective graphene at ≈85%. The reported polymer-assisted RTA defect healing method shows promise for healing other 2D materials with other dopants by simply changing the chemistry of the polymeric nanobandage.

Graphene-Enabled High-Performance Electrokinetic Focusing and Sensing.

Transverse isoelectric focusing, i.e., isoelectric focusing that is normal to the fluid-flow direction, is an electrokinetic method ideal for micro total analysis. However, a major challenge remains: There is no electrode system integrable in a microfluidic device to allow reliable transverse isoelectric focusing and electrokinetic sensing. Here, we overcome this barrier by developing devices that incorporate microelectrodes made of monolayer graphene. We find that the electrolysis stability over time for graphene microelectrodes is >103× improved compared to typical microfabricated inert-metal microelectrodes. Through transverse isoelectric focusing between graphene microelectrodes, within minutes, specific proteins can be separated and concentrated to scales of ∼100 µm. Based on the concentrating effect and the high optical transparency of graphene, we develop a three-dimensional multistream microfluidic strategy for label-free detection of the proteins at same processing position with a sensitivity that is ∼102× higher than those of the state-of-the-art label-free sensors. These results demonstrate the advantage of monolayer-graphene microelectrodes for high-performance electrokinetic analysis to allow lab-on-a-chips of maximal time and size efficiencies.

Flow-sensory contact electrification of graphene.

All-electronic interrogation of biofluid flow velocity by electrical nanosensors incorporated in ultra-low-power or self-sustained systems offers the promise of enabling multifarious emerging research and applications. However, existing nano-based electrical flow sensing technologies remain lacking in precision and stability and are typically only applicable to simple aqueous solutions or liquid/gas dual-phase mixtures, making them unsuitable for monitoring low-flow (~micrometer/second) yet important characteristics of continuous biofluids (such as hemorheological behaviors in microcirculation). Here, we show that monolayer-graphene single microelectrodes harvesting charge from continuous aqueous flow provide an effective flow sensing strategy that delivers key performance metrics orders of magnitude higher than other electrical approaches. In particular, over six-months stability and sub-micrometer/second resolution in real-time quantification of whole-blood flows with multiscale amplitude-temporal characteristics are obtained in a microfluidic chip.

Characterization of an engineered water-soluble variant of the full-length human mu opioid receptor.

A water-soluble variant of the transmembrane domain of the human mu opioid receptor (wsMOR-TM) was previously characterized. This study explored whether the full-length version of the engineered water-soluble receptor, (wsMOR-FL), could be overexpressed in Escherichia coli and if it would retain water solubility, binding capability and thermostability. wsMOR was over-expressed and purified in E. coli BL21(DE3) cells (EMD/Novagen) as we reported previously for the wsMOR-TM. Both native N and C termini were added back to the highly engineered wsMOR-TM. Six His-tag was added in the N terminus for purification purposes. The wsMOR-FL was characterized using atomic force microscope for its monomeric state, circular dichroism for its secondary structure and thermostability. Its binding with naltrexone is also determined. Compared to the native human MOR, wsMOR-FL displays similar helical secondary structure content and comparable affinity (nM) for the antagonist naltrexone. The secondary structure of the receptor remains stable within a wide range of pH (6-9). In contrast to the transmembrane portion, the secondary structure of full-length receptor tolerated a wide range of temperature (10-90 °C). The receptor remains predominantly as a monomer in solution, as directly imaged using atomic force microscopy. This study demonstrated that functional full-length water-soluble variant of human mu receptor can be over-expressed and purified using an E. coli over-expression system. This provides a novel tool for the investigation of structural and functional properties of the human MOR. N- and C-termini strengthened the thermostability of the protein in this specific water soluble variant. Communicated by Ramaswamy H. Sarma.

Scalable Arrays of Chemical Vapor Sensors Based on DNA-Decorated Graphene.

Arrays of DNA-functionalized graphene field-effect transistors (gFETs) hold great promise for high-performance vapor sensing. In this chapter, we describe methods for the scalable production of gFET-based vapor sensors with high sensitivity and efficiency in size, cost, and time. Large-area graphene sheets were prepared via chemical vapor deposition (CVD); a standard photolithographic processing for large-area graphene was used to fabricate gFETs with high mobility and low doping level under ambient conditions. The gFETs were functionalized by single-stranded DNA (ssDNA), which binds to the graphene channels through π-π stacking interaction and provides affinity to a wide range of chemical vapors. The resulting sensing arrays demonstrate detection of target vapor molecules down to parts-per-million concentrations with high selectivity among analytes with high chemical similarity including a series of carboxylic acids and structural isomers of carboxylic acids and pinene.

DNA Nanotweezers and Graphene Transistor Enable Label-Free Genotyping.

Electronic DNA-biosensor with a single nucleotide resolution capability is highly desirable for personalized medicine. However, existing DNA-biosensors, especially single nucleotide polymorphism (SNP) detection systems, have poor sensitivity and specificity and lack real-time wireless data transmission. DNA-tweezers with graphene field effect transistor (FET) are used for SNP detection and data are transmitted wirelessly for analysis. Picomolar sensitivity of quantitative SNP detection is achieved by observing changes in Dirac point shift and resistance change. The use of DNA-tweezers probe with high-quality graphene FET significantly improves analytical characteristics of SNP detection by enhancing the sensitivity more than 1000-fold in comparison to previous work. The electrical signal resulting from resistance changes triggered by DNA strand-displacement and related changes in the DNA geometry is recorded and transmitted remotely to personal electronics. Practical implementation of this enabling technology will provide cheaper, faster, and portable point-of-care molecular health status monitoring and diagnostic devices.

Detection of Sub-fM DNA with Target Recycling and Self-Assembly Amplification on Graphene Field-Effect Biosensors.

All-electronic DNA biosensors based on graphene field-effect transistors (GFETs) offer the prospect of simple and cost-effective diagnostics. For GFET sensors based on complementary probe DNA, the sensitivity is limited by the binding affinity of the target oligonucleotide, in the nM range for 20 mer targets. We report a ∼20 000× improvement in sensitivity through the use of engineered hairpin probe DNA that allows for target recycling and hybridization chain reaction. This enables detection of 21 mer target DNA at sub-fM concentration and provides superior specificity against single-base mismatched oligomers. The work is based on a scalable fabrication process for biosensor arrays that is suitable for multiplexed detection. This approach overcomes the binding-affinity-dependent sensitivity of nucleic acid biosensors and offers a pathway toward multiplexed and label-free nucleic acid testing with high accuracy and selectivity.

All-Electronic Quantification of Neuropeptide-Receptor Interaction Using a Bias-Free Functionalized Graphene Microelectrode.

Opioid neuropeptides play a significant role in pain perception, appetite regulation, sleep, memory, and learning. Advances in understanding of opioid peptide physiology are held back by the lack of methodologies for real-time quantification of affinities and kinetics of the opioid neuropeptide-receptor interaction at levels typical of endogenous secretion (<50 pM) in biosolutions with physiological ionic strength. To address this challenge, we developed all-electronic opioid-neuropeptide biosensors based on graphene microelectrodes functionalized with a computationally redesigned water-soluble µ-opioid receptor. We used the functionalized microelectrode in a bias-free charge measurement configuration to measure the binding kinetics and equilibrium binding properties of the engineered receptor with [d-Ala2, N-MePhe4, Gly-ol]-enkephalin and ß-endorphin at picomolar levels in real time.

Crystalline Bilayer Graphene with Preferential Stacking from Ni-Cu Gradient Alloy.

We developed a high-yield synthesis of highly crystalline bilayer graphene (BLG) with two preferential stacking modes using a Ni-Cu gradient alloy growth substrate. Previously reported approaches for BLG growth include flat growth substrates of Cu or Ni-Cu uniform alloys and "copper pocket" structures. Use of flat substrates has the advantage of being scalable, but the growth mechanism is either "surface limited" (for Cu) or carbon precipitation (for uniform Ni-Cu), which results in multicrystalline BLG grains. For copper pockets, growth proceeds through a carbon back-diffusion mechanism, which leads to the formation of highly crystalline BLG, but scaling of the copper pocket structure is expected to be difficult. Here we demonstrate a Ni-Cu gradient alloy that combines the advantages of these earlier methods: the substrate is flat, so easy to scale, while growth proceeds by a carbon back-diffusion mechanism leading to high-yield growth of BLG with high crystallinity. The BLG layer stacking was almost exclusively Bernal or twisted with an angle of 30°, consistent with first-principles calculations we conducted. Furthermore, we demonstrated scalable production of transistor arrays based crystalline Bernal-stacked BLG with a band gap that was tunable at room temperature.

Structural-functional analysis of engineered protein-nanoparticle assemblies using graphene microelectrodes.

The characterization of protein-nanoparticle assemblies in solution remains a challenge. We demonstrate a technique based on a graphene microelectrode for structural-functional analysis of model systems composed of nanoparticles enclosed in open-pore and closed-pore ferritin molecules. The method readily resolves the difference in accessibility of the enclosed nanoparticle for charge transfer and offers the prospect for quantitative analysis of pore-mediated transport, while shedding light on the spatial orientation of the protein subunits on the nanoparticle surface, faster and with higher sensitivity than conventional catalysis methods.

An Aptamer-Based Biosensor for the Azole Class of Antifungal Drugs.

This technical report describes the development of an aptamer for sensing azole antifungal drugs during therapeutic drug monitoring. Modified synthetic evolution of ligands through exponential enrichment (SELEX) was used to discover a DNA aptamer recognizing azole class antifungal drugs. This aptamer undergoes a secondary structural change upon binding to its target molecule, as shown through fluorescence anisotropy-based binding measurements. Experiments using circular dichroism spectroscopy revealed a unique G-quadruplex structure that was essential and specific for binding to the azole antifungal target. Aptamer-functionalized graphene field effect transistor (GFET) devices were created and used to measure the strength of binding of azole antifungals to this surface. In total, this aptamer and the supporting sensing platform provide a valuable tool for therapeutic drug monitoring of patients with invasive fungal infections. IMPORTANCE We have developed the first aptamer directed toward the azole class of antifungal drugs and a functional biosensor for these drugs. This aptamer has a unique secondary structure that allows it to bind to highly hydrophobic drugs. The aptamer works as a capture component of a graphene field effect transistor device. These devices can provide a quick and easy assay for determining drug concentrations. These will be useful for therapeutic drug monitoring of azole antifungal drugs, which is necessary to deal with the complex drug dosage profiles.

pH Sensing Properties of Flexible, Bias-Free Graphene Microelectrodes in Complex Fluids: From Phosphate Buffer Solution to Human Serum.

Advances in techniques for monitoring pH in complex fluids can have a significant impact on analytical and biomedical applications. This study develops flexible graphene microelectrodes (GEs) for rapid (<5 s), very-low-power (femtowatt) detection of the pH of complex biofluids by measuring real-time Faradaic charge transfer between the GE and a solution at zero electrical bias. For an idealized sample of phosphate buffer solution (PBS), the Faradaic current is varied monotonically and systematically with the pH, with a resolution of ≈0.2 pH unit. The current-pH dependence is well described by a hybrid analytical-computational model, where the electric double layer derives from an intrinsic, pH-independent (positive) charge associated with the graphene-water interface and ionizable (negative) charged groups. For ferritin solution, the relative Faradaic current, defined as the difference between the measured current response and a baseline response due to PBS, shows a strong signal associated with ferritin disassembly and the release of ferric ions at pH ≈2.0. For samples of human serum, the Faradaic current shows a reproducible rapid (<20 s) response to pH. By combining the Faradaic current and real-time current variation, the methodology is potentially suitable for use to detect tumor-induced changes in extracellular pH.

Quantifying the effect of ionic screening with protein-decorated graphene transistors.

Liquid-based applications of biomolecule-decorated field-effect transistors (FETs) range from biosensors to in vivo implants. A critical scientific challenge is to develop a quantitative understanding of the gating effect of charged biomolecules in ionic solution and how this influences the readout of the FETs. To address this issue, we fabricated protein-decorated graphene FETs and measured their electrical properties, specifically the shift in Dirac voltage, in solutions of varying ionic strength. We found excellent quantitative agreement with a model that accounts for both the graphene polarization charge and ionic screening of ions adsorbed on the graphene as well as charged amino acids associated with the immobilized protein. The technique and analysis presented here directly couple the charging status of bound biomolecules to readout of liquid-phase FETs fabricated with graphene or other two-dimensional materials.

Scalable Production of Sensor Arrays Based on High-Mobility Hybrid Graphene Field Effect Transistors.

We have developed a scalable fabrication process for the production of DNA biosensors based on gold nanoparticle-decorated graphene field effect transistors (AuNP-Gr-FETs), where monodisperse AuNPs are created through physical vapor deposition followed by thermal annealing. The FETs are created in a four-probe configuration, using an optimized bilayer photolithography process that yields chemically clean devices, as confirmed by XPS and AFM, with high carrier mobility (3590 ± 710 cm2/V·s) and low unintended doping (Dirac voltages of 9.4 ± 2.7 V). The AuNP-Gr-FETs were readily functionalized with thiolated probe DNA to yield DNA biosensors with a detection limit of 1 nM and high specificity against noncomplementary DNA. Our work provides a pathway toward the scalable fabrication of high-performance AuNP-Gr-FET devices for label-free nucleic acid testing in a realistic clinical setting.

Scalable Production of High-Sensitivity, Label-Free DNA Biosensors Based on Back-Gated Graphene Field Effect Transistors.

Scalable production of all-electronic DNA biosensors with high sensitivity and selectivity is a critical enabling step for research and applications associated with detection of DNA hybridization. We have developed a scalable and very reproducible (>90% yield) fabrication process for label-free DNA biosensors based upon graphene field effect transistors (GFETs) functionalized with single-stranded probe DNA. The shift of the GFET sensor Dirac point voltage varied systematically with the concentration of target DNA. The biosensors demonstrated a broad analytical range and limit of detection of 1 fM for 60-mer DNA oligonucleotide. In control experiments with mismatched DNA oligomers, the impact of the mismatch position on the DNA hybridization strength was confirmed. This class of highly sensitive DNA biosensors offers the prospect of detection of DNA hybridization and sequencing in a rapid, inexpensive, and accurate way.

Monolayer Single-Crystal 1T'-MoTe2 Grown by Chemical Vapor Deposition Exhibits Weak Antilocalization Effect.

Growth of transition metal dichalcogenide (TMD) monolayers is of interest due to their unique electrical and optical properties. Films in the 2H and 1T phases have been widely studied but monolayers of some 1T'-TMDs are predicted to be large-gap quantum spin Hall insulators, suitable for innovative transistor structures that can be switched via a topological phase transition rather than conventional carrier depletion [ Qian et al. Science 2014 , 346 , 1344 - 1347 ]. Here we detail a reproducible method for chemical vapor deposition of monolayer, single-crystal flakes of 1T'-MoTe2. Atomic force microscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, and transmission electron microscopy confirm the composition and structure of MoTe2 flakes. Variable temperature magnetotransport shows weak antilocalization at low temperatures, an effect seen in topological insulators and evidence of strong spin-orbit coupling. Our approach provides a pathway to systematic investigation of monolayer, single-crystal 1T'-MoTe2 and implementation in next-generation nanoelectronic devices.

Seeded growth of highly crystalline molybdenum disulphide monolayers at controlled locations.

Monolayer transition metal dichalcogenides are materials with an atomic structure complementary to graphene but diverse properties, including direct energy bandgaps, which makes them intriguing candidates for optoelectronic devices. Various approaches have been demonstrated for the growth of molybdenum disulphide (MoS2) on insulating substrates, but to date, growth of isolated crystalline flakes has been demonstrated at random locations only. Here we use patterned seeds of molybdenum source material to grow flakes of MoS2 at predetermined locations with micrometre-scale resolution. MoS2 flakes are predominantly monolayers with high material quality, as confirmed by atomic force microscopy, transmission electron microscopy and Raman and photoluminescence spectroscopy. As the monolayer flakes are isolated at predetermined locations, transistor fabrication requires only a single lithographic step. Device measurements exhibit carrier mobility and on/off ratio that exceed 10 cm(2) V(-1) s(-1) and 10(6), respectively. The technique provides a path for in-depth physical analysis of monolayer MoS2 and fabrication of MoS2-based integrated circuits.

Disorder-induced magnetoresistance in a two-dimensional electron system.

We predict and demonstrate that a disorder-induced carrier density inhomogeneity causes magnetoresistance (MR) in a two-dimensional electron system. Our experiments on graphene show a quadratic MR persisting far from the charge neutrality point. Effective medium calculations show that for charged impurity disorder, the low-field MR is a universal function of the ratio of carrier density to fluctuations in carrier density, a power law when this ratio is large, in excellent agreement with experiment. The MR is generic and should occur in other materials with large carrier density inhomogeneity.