Identification of RNA-based cell-type markers for stem-cell manufacturing systems with a statistical scoring function.

Cell-type biomarkers are useful in stem-cell manufacturing to monitor cell purity, quantity, and quality. However, the study on cell-type markers, specifically for stem cell manufacture, is limited. Emerging questions include which RNA transcripts can serve as biomarkers during stem cell culture and how to discover these biomarkers efficiently and precisely. We developed a scoring function system to identify RNA biomarkers with RNA-seq data for systems that have a limited number of cell types. We applied the method to two data sets, one for extracellular RNAs (ex-RNAs) and the other for intracellular microRNAs (miRNAs). The first data set has RNA-seq data of ex-RNAs from cell culture media for six different types of cells, including human embryonic stem cells. To get the RNA-seq data from intracellular miRNAs, we cultured three types of cells: human embryonic stem cells (H9), neural stem cells (NSC), hESC-derived endothelial cells (EC) and conducted small RNA-seq to their intracellular miRNAs. Using these data, we identified a set of ex-RNAs/smRNAs as candidates of biomarkers for different types of cells for cell manufacture. The validity of these findings was confirmed by the utilization of additional data sets and experimental procedures. We also used deep-learning-based prediction methods and simulated data to validate these discovered biomarkers.

Electrochemical Beacon Method to Quantify 10 Attomolar Nucleic Acids with a Semilog Dynamic Range of 7 Orders of Magnitude.

Change in the dynamics of single-stranded DNA or RNA probes tethered to an Au electrode on immunospecific binding to the analyte is a versatile approach to quantify a variety of molecules, such as heavy metal ions, pesticides, proteins, and nucleic acids (NAs). A widely studied approach is the electrochemical beacon method where the redox of a dye attached to the probe decreases as its proximity to the underlying electrode changes on binding. The limit of quantification (LOQ) defined by the semilog dependence of the signal on target concentration is in the picomolar range. Here, a method was studied where, by differential reflectivity, multiple reactions were measured on a monolith electrode. An alternative contrast mechanism was discovered, which led to an approach to enhance the LOQ to 10 aM and increase the dynamic range to 7 orders of magnitude using similar probes and binding conditions. Quantitative analysis on sequences with the G-C fraction ranging from 37 to 72% was performed. The approach will allow for the development of a label-free, enzyme-free microarray to detect biomolecules including NAs and proteins on a single electrode at quantification from 10 aM to 0.1 nM with high specificity.

Quantitative PCR of Small Nucleic Acids: Size Matters.

Quantitative dysregulation in small nucleic acids (NA), such as microRNA (miRNA), extracted from minimally invasive biopsies, such as, blood, stool, urine, nose, throat, are promising biomarker for diseases diagnosis and management. We quantify the effect of the extra step of poly(A) ligation for cDNA synthesis and small size of the NA on the limit of quantification (LOQ) of quantitative PCR (qPCR), the gold standard to measure copy number. It was discovered that for small NA, the cycle threshold, Ct that is proportional to -log[c], where [c] is the concentration of the target NA exhibits a sharp transition. The results indicate that although the limit of detection (LOD) of qPCR can be in femtomolar range, the LOQ is significantly reduced by well over three orders of magnitude, in picomolar range. Specifically, the study reveals that the PCR product length is the primary reason the limitation on LOQ and is explicitly shown to be an important consideration for primer design for qPCR in general.

Electrochemical Deposition of Polyelectrolytes Is Maximum at the Potential of Zero Charge.

Electrochemical deposition of cationic and anionic polyelectrolyte on a Au electrode is studied as a function of applied potential between the electrode and the solution of monovalent electrolyte. The deposition is measured by open circuit potential relative to a pristine electrode in a reference solution (100 mM NaCl). The rate of deposition is measured by a home-built electrochemical-optical method in real time. It was discovered that the polarity of the potential and magnitude of the potential are not the primary reasons to enhance deposition. For example, both the amount and rate of deposition of negatively charged poly(styrenesulfonate) in NaCl are higher when the electrode is at -200 mV than at +200 mV with respect to the solution. The results are explained in terms of the charge state of the electrical double layer that is primarily controlled by supporting (small) ions.

Electrochemical Characteristics of a DNA Modified Electrode as a Function of Percent Binding.

Electrochemical characteristics of immobilized double-stranded DNA (dsDNA) on a Au electrode were studied as a function of coverage using a home-built optoelectrochemical method. The method allows probing of local redox processes on a 6 µm spot by measuring both differential reflectivity (SEED-R) and interferometry (SEED-I). The former is sensitive to redox ions that tend to adsorb to the electrode, while SEED-I is sensitive to nonadsorbing ions. The redox reaction maxima, Rmax and Δmax from SEED-R and SEED-I, respectively, are linearly proportional to amperometric peak current, Imax. The DNA binding is measured by a redox active dye, methylene blue, that intercalates in dsDNA, leading to an Rmax. Concomitantly, the absence of Δmax for [Fe(CN)6]4-/3- by SEED-I ensures that there is no leakage current from voids/defects in the alkanethiol passivation layer at the same spot of measurement. The binding was regulated electrochemically to obtain the binding fraction, f, ranging about three orders of magnitude. A remarkably sharp transition, f = fT = 1.25 × 10-3, was observed. Below fT, dsDNA molecules behaved as individual single-molecule nanoelectrodes. Above the crossover transition, Rmax, per dsDNA molecule dropped rapidly as f-1/2 toward a planar-like monolayer. The SEED-R peak at f ∼ 3.3 × 10-4 (∼270 dsDNA molecules) was (statistically) robust, corresponding to a responsivity of ∼0.45 zeptomoles of dsDNA/spot. Differential pulse voltammetry in the single-molecule regime estimated that the current per dsDNA molecule was ∼4.1 fA. Compared with published amperometric results, the reported semilogarithmic dependence on target concentration is in the f > fT regime.

Simultaneous Printing of Two Inks by Contact Lithography.

Microcontact printing (µCP) is a valuable technique used to fabricate complex patterns on surfaces for applications such as sensors, cell seeding, self-assembled monolayers of proteins and nanoparticles, and micromachining. The process is very precise but is typically confined to depositing a single type of ink per print, which limits the complexity of using multifunctionality patterns. Here we describe a process by which two inks are printed concomitantly in a single operation to create an alternating pattern of hydrophobic and hydrophilic characteristics. The hydrophobic ink, PDMS, is deposited by evaporation on the noncontact region, while the hydrophilic polyelectrolyte is transferred on contact. We demonstrate that there is no gap between the two patterns using an optical-electrochemical method. We describe some potential applications of this method, including layer-by-layer deposition of polyelectrolytes for sensors and creation of a scaffold for cell culture.

Quantitative Electrochemical DNA Microarray on a Monolith Electrode with Ten Attomolar Sensitivity, 100% Specificity, and Zero Background.

Circulating microRNA are promising diagnostic and prognostic biomarkers of disease in quantitative blood tests. A label-free, PCR-free, electrochemical microarray technology on a monolith electrode is described, with 10 attomolar (aM) sensitivity and responsiveness to binding of <1 zeptomole of target to immobilized ssDNA probes with zero background. Specificity is 100% in a mixture with five nonspecific miRNA each with a 103-fold higher concentration. Direct measurement on plasma-derived miRNA without cDNA conversion and PCR demonstrated multiplexing and near-ideal quantitative correlation with an equivalent pure sample. The dynamic range is a target concentration ranging from 10-2 to 103 femtomolar (fM). This PCR-free novel technology can be applied as a test for cancer diagnosis/prognosis to detect 103 copies of a miRNA sequence in RNA extracted from 100 µL of plasma.

Noninvasive Measurement of Electrical Events Associated with a Single Chlorovirus Infection of a Microalgal Cell.

Chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) contains a viral-encoded K(+) channel imbedded in its internal membrane, which triggers host plasma membrane depolarization during virus infection. This early stage of infection was monitored at high resolution by recording the cell membrane depolarization of a single Chlorella cell during infection by a single PBCV-1 particle. The measurement was achieved by depositing the cells onto a network of one-dimensional necklaces of Au nanoparticles, which spanned two electrodes 70 µm apart. The nanoparticle necklace array has been shown to behave as a single-electron device at room temperature. The resulting electrochemical field-effect transistor (eFET) was gated by the cell membrane potential, which allowed a quantitative measurement of the electrophysiological changes across the rigid cell wall of the microalgae due to a single viral attack at high sensitivity. The single viral infection signature was quantitatively confirmed by coupling the eFET measurement with a method in which a single viral particle was delivered for infection by a scanning probe microscope cantilever.

Negative printing by soft lithography.

In inkless microcontact printing (IµCP) by soft lithography, the poly(dimethylsiloxane) (PDMS) stamp transfers uncured polymer to a substrate corresponding to its pattern. The spontaneous diffusion of PDMS oligomers to the surface of the stamp that gives rise to this deleterious side effect has been leveraged to fabricate a variety of devices, such as organic thin film transistors, single-electron devices, and biomolecular chips. Here we report an anomalous observation on a partially cured PDMS stamp where the transfer of oligomers onto Au occurred on regions that were not in contact with the stamp, while the surface in contact with the stamp was pristine with no polymer. On the SiO2 surface of the same chip, as expected, the transfer of PDMS occurred exclusively on regions in contact with the stamp. The printing on Au was quantified by a novel method where the submonolayer of PDMS transfer was measured by probing the local electrochemical passivation of the Au. The local transfer of polymer on SiO2 (and also Au) was measured by selective deposition of Au nanoparticle necklaces that exclusively deposited on PDMS at submonolayer sensitivity. It was discovered that the selectivity and sharpness of PDMS deposition on Au for inkless printing (i.e., negative) is significantly better than the traditional (positive) microcontact printing where the stamp is "inked" with low molecular weight PDMS.

Tactile imaging of an imbedded palpable structure for breast cancer screening.

Apart from texture, the human finger can sense palpation. The detection of an imbedded structure is a fine balance between the relative stiffness of the matrix, the object, and the device. If the device is too soft, its high responsiveness will limit the depth to which the imbedded structure can be detected. The sensation of palpation is an effective procedure for a physician to examine irregularities. In a clinical breast examination (CBE), by pressing over 1 cm(2) area, at a contact pressure in the 70-90 kPa range, the physician feels cancerous lumps that are 8- to 18-fold stiffer than surrounding tissue. Early detection of a lump in the 5-10 mm range leads to an excellent prognosis. We describe a thin-film tactile device that emulates human touch to quantify CBE by imaging the size and shape of 5-10 mm objects at 20 mm depth in a breast model using ∼80 kPa pressure. The linear response of the device allows quantification where the greyscale corresponds to the relative local stiffness. The (background) signal from <2.5-fold stiffer objects at a size below 2 mm is minimal.

Multianalyte electrochemical biosensor on a monolith electrode by optically scanning the electrical double layer.

Redox on an electrode is an interfacial phenomenon that modulates the charge in the electrical double layer (EDL). A novel instrument, the Scanning Electrometer for Electrical Double-layer (SEED) has been developed to measure multiple enzyme reactions on a monolith electrode due to immunospecific binding with a mixture of respective analytes. SEED quantitatively maps the local redox reaction by scanning a laser on the array of enzyme monolayer spots immobilized on the monolith electrode. SEED measures the change in local charge state of the EDL that abruptly changes due to the redox reaction. The measurement spot size defined by the size of the laser beam is ~10 µm. The SEED signal is linearly proportional to the local redox current density and analyte concentration. The specificity is close to 100%. The SEED readout is compatible with microfluidics platform where the signal degrades less than 2% due to the poly(dimethyl siloxane) (PDMS) body.

Noninvasive measurement of membrane potential modulation in microorganisms: photosynthesis in green algae.

Cell membrane potential (CMP) modulation is a physical measurement to quantitatively probe cell physiology in real time at high specificity. Electrochemical field effect transistors (eFETs) made from graphene and Si nanowire provide strong mechanical and electrical coupling with neurons and muscle cells to noninvasively measure CMP at high sensitivity. To date, there are no noninvasive methods to study electrophysiology of microorganisms because of stiff cell walls and significantly smaller membrane polarizations. An eFET made from the smallest possible nanostructure, a nanoparticle, with sensitivity to a single-electron charge is developed to noninvasively measure CMP modulation in algae. The applicability of the device is demonstrated by measuring CMP modulation due to a light-induced proton gradient inside the chloroplast during photosynthesis. The ∼9 mV modulation in CMP in algae is consistent with the absorbance spectrum of chlorophyll, photosynthetic pathway, and inorganic carbon source concentration in the environment. The method can potentially become a routine method to noninvasively study electrophysiology of cells, such as microorganisms for biofuels.

Self-assembly of gold nanoparticles on poly(allylamine hydrochloride) nanofiber: a new route to fabricate "necklace" as single electron devices.

Fabrication of a percolating conductive device exhibiting "necklace-like morphology" is being reported utilizing a new route. This device was fabricated by exploiting the electrostatic self-assembly of citrate capped negatively charged Au nanoparticles (NPs) (60 nm diameter) over positively charged poly(allylamine hydrochloride) (PAH) fibrous scaffold and followed by synthesis of small Au NPs (∼10 nm) on the PAH surface. These 10 nm Au NPs were selectively synthesized over the PAH fiber surface using the surface catalyzed reduction of Au precursor (HAuCl4), leading to a continuous conducting network. This conducting device demonstrated a room temperature (RT) Coulomb-blockade characteristic, which is indicative of "single electron device". The deposition of Au NPs was directed by the diameter of PAH fibers and UV-irradiation exposure time used during the synthesis process. The average diameter of the fibers was in the ∼100-150 nm range, and the polyelectrolyte (PAH) was fabricated using the electrospinning technique. The size of these fibers was controlled by tuning the physical properties of PAH solution. Exposure of UV-irradiation for 25 min was sufficiently enough to deposit Au NPs in close proximity to each other. Longer exposure time (∼60 min) resulted in a device which showed linear Ohmic current-voltage (I-V) behavior. The present process is reproducible, efficient, and resulted in a structurally stable and robust device.

Imaging electroluminescence from individual nanoparticles in an array exhibiting room temperature single electron effect.

Electroluminescence (EL) from the monolayer of a network of a one-dimensional (1D) necklace of 10 nm Au particles (nano)cemented by CdS is imaged. The EL and photoluminescence (PL) spectra confirm the emission from CdS. The EL emission blinks and is highly specular. The position of the speckles from individual CdS cement sites is independent of magnitude and polarity of the applied bias. The EL is explained by field-assisted ionization of the cement due to high internal fields in the array caused by stationary local charging that also leads to robust single electron effect at room temperature.

Single electron transistor in aqueous media.

A gold nanoparticle necklace array spanning a ∼30-micrometer-wide channel shows a robust coulomb blockade effect at room temperature with a threshold of 1V in air. When this device is operated in the aqueous solution, a gain of ∼130 fold in conductance is obtained in electrochemical gating, significantly higher than other nanomaterial-based electrochemical transistors.

Direct mapping of local redox current density on a monolith electrode by laser scanning.

An optical method of mapping local redox reaction over a monolith electrode using simple laser scanning is described. As the optical signal is linearly proportional to the maximum redox current that is measured concomitantly by voltammetry, the optical signal quantitatively maps the local redox current density distribution. The method is demonstrated on two types of reactions: (1) a reversible reaction where the redox moieties are ionic, and (2) an irreversible reaction on two different types of enzymes immobilized on the electrode where the reaction moieties are nonionic. To demonstrate the scanning capability, the local redox behavior on a "V-shaped" electrode is studied where the local length scale and, hence, the local current density, is nonuniform. The ability to measure the current density distribution by this method will pave the way for multianalyte analysis on a monolith electrode using a standard three-electrode configuration. The method is called Scanning Electrometer for Electrical Double-layer (SEED).

Ultrasoft 100 nm thick zero Poisson's ratio film with 60% reversible compressibility.

About a 100 nm thick multilayer film of nanoparticle monolayers and polymer layers is shown to behave like cellular-foam with a modulus below 100 KPa. The 1.25 cm radius film adhered to a rigid surface can be compressed reversibly to 60% strain. The more than 4 orders of magnitude lower modulus compared to its constituents is explained by considering local bending in the (nano)cellular structure, similar to cork and wings of beetles. As the rigidity of the polymer backbone is increased in just four monolayers, the modulus of the composite increases by over 70%. Electro-optical map of the strain distribution over the area of compression and increase in modulus with thickness indicates the films have zero Poisson's ratio.

Tuning the energy level offset between donor and acceptor with ferroelectric dipole layers for increased efficiency in bilayer organic photovoltaic cells.

Ultrathin ferroelectric polyvinylidene fluoride (70%)-tetrafluoroethylene (30%) copolymer film is inserted between the poly3(hexylthiophene) (P3HT) donor and [6,6]-phenyl-C61-butyric acid methylester (PCBM) acceptor layers as the dipole layer to tune the relative energy levels, which can potentially maximize the open circuit voltage of bilayer organic solar cells. In this work, the power conversion efficiency of P3HT/PCBM bilayer solar cells is demonstrated to be doubled with the inserted dipoles.

Modulation of electron tunneling in a nanoparticle array by sound waves: an avenue to high-speed, high-sensitivity sensors.

Using an electrostatic self-assembly process, metal nanoparticles are deposited on polyelectrolyte fibers such that the interparticle distance between the nanoparticles is comparable to the polyelectrolyte's molecular width. By modulating the dielectric properties of the interparticle polymer layer, a highly sensitive, reversible humidity sensor with an ultrafast response time of ≈3 ms is demonstrated. The higher sensitivity at low humidity shows a conductivity increase by over two orders of magnitude in response to a change in relative humidity from 21 to 1%.