Leyla Soleymani

Programming the detection limits of biosensors through controlled nanostructuring

Advances in materials chemistry offer a range of nanostructured shapes and textures for building new biosensors. Previous reports have implied that controlling the properties of sensor substrates can improve detection sensitivities, but the evidence remains indirect. Here we show that by nanostructuring the sensing electrodes, it is possible to create nucleic acid sensors that have a broad range of sensitivities and that are capable of rapid analysis. Only highly branched electrodes with fine structuring attained attomolar sensitivity. Nucleic acid probes immobilized on finely nanostructured electrodes appear more accessible and therefore complex more rapidly with target molecules in solution. By forming arrays of microelectrodes with different degrees of nanostructuring, we expanded the dynamic range of a sensor system from two to six orders of magnitude. The demonstration of an intimate link between nanoscale sensor structure and biodetection sensitivity will aid the development of high performance diagnostic tools for biology and medicine.

Direct, Electronic MicroRNA Detection for the Rapid Determination of Differential Expression Profiles

cellular levels of miRNAs is challenging, however, because their short lengths, low abundances, and high levels of sequence similarity present obstacles in the use of conventional analytical methods. Hybridization-based approaches (e.g. microarray analyses) are attractive for microRNA profiling because of the potential for extensive multiplexing and the discrimination of closely related sequences; however, such methodology requires large quantities (micrograms) of starting material. [6–8] The lack of sensitivity of existing arraybased methods is related to the type of readout used: typically fluorescence signals emitted from an RNA-conjugated fluorophore. Very low levels of signal derived from low-abundance sequences are extremely difficult to detect without sophisticated optics. Impressive progress in this area has been made with the development of novel methods for the ultrasensitive detection of miRNA hybridization on array surfaces; [9, 10] however, the methods available involve many steps and have not yet been validated with biological samples. We describe herein a new approach to ultrasensitive, direct, hybridization-based microRNA profiling using a multiplexed electronic chip and electrocatalytic readout. The very high sensitivity of this method enables the direct analysis of small samples (nanograms of total RNA) within 30 minutes. The power of this method is demonstrated by the identification of specific microRNA sequences that are overexpressed in human head and neck cancer cells relative to normal epithelial cells. We endeavored to develop a new method for microRNA profiling that would feature the convenience of array-based analysis, but would augment the power of such multiplexing with the exceptional sensitivity required to assay small biological samples for low-abundance microRNAs. We pursued an approach based on electronic readout and prepared a

Hierarchical Nanotextured Microelectrodes Overcome the Molecular Transport Barrier To Achieve Rapid, Direct Bacterial Detection

Detection of biomolecules at low abundances is crucial to the rapid diagnosis of disease. Impressive sensitivities, typically measured with small model analytes, have been obtained with a variety of nano- and microscale sensors. A remaining challenge, however, is the rapid detection of large native biomolecules in real biological samples. Here we develop and investigate a sensor system that directly addresses the source of this challenge: the slow diffusion of large biomolecules traveling through solution to fixed sensors, and inefficient complexation of target molecules with immobilized probes. We engineer arrayed sensors on two distinct length scales: a ∼100 μm length scale commensurable with the distance bacterial mRNA can travel in the 30 min sample-to-answer duration urgently required in point-of-need diagnostic applications; and the nanometer length scale we prove necessary for efficient target capture. We challenge the specificity of our hierarchical nanotextured microsensors using crude bacterial lysates and document the first electronic chip to sense trace levels of bacteria in under 30 min.

Nanostructuring of Patterned Microelectrodes To Enhance the Sensitivity of Electrochemical Nucleic Acids Detection

Disease diagnosis on the basis of biomolecular analysis requires sensitive, cost-effective, and multiplexed assays. Biomarker analysis based on electronic readout has long been cited as a promising approach that would enable the creation of a new family of chip-based devices with appropriate cost and sensitivity for medical testing. The sensitivity of electronic readout, and specifically electrochemical analysis, is in principle sufficient to enable direct detection of small numbers of analyte molecules with simple instrumentation. Over the last several years, very high sensitivities have been demonstrated for nanomaterial-based electrochemical assays in particular, whereby nanowireand nanotube-based electrodes have shown some of the highest sensitivities to date. Whether these assays can be made practical and multiplexed remains to be seen, however, as the materials used have not been readily amenable to arrayed and straightforward fabrication. Herein, we present a new system that enables nanostructured materials to be produced and used as nucleic acids sensors in an arrayed format. By using lithographically defined apertures as a template, we grew microelectrodes on a silicon chip by metal electrodeposition (Figure 1). Drawing upon the numerous studies of nanostructures with diverse morphologies generated as dispersions in solution, we sought to manipulate precisely the surface morphology of these electrodes to control the level of nanostructuring present. We show that the production of nanostructured features on electrode surfaces is essential for the performance of the microelectrodes as ultrasensitive electrochemical detectors. A variety of studies have suggested that nanostructures are highly beneficial for biosensing applications because of the increased surface area, enhanced delivery of amplification agents, or precise biomolecule–electrode connections that are possible; however, the role of nano-

Direct profiling of cancer biomarkers in tumor tissue using a multiplexed nanostructured microelectrode integrated circuit.

The analysis of panels of nucleic acid biomarkers offers valuable diagnostic and prognostic information for cancer management. A cost-effective, highly sensitive electronic chip would offer an ideal platform for clinical biomarker readout and would have maximal utility if it was (i) multiplexed, enabling on-chip assays of multiple biomarkers, and (ii) able to perform direct (PCR-free) readout of disease-related genes. Here we report a chip onto which we integrate novel nanostructured microelectrodes and with which we directly detect cancer biomarkers in heterogeneous biological samples-both cell extracts and tumor tissues. Coarse photolithographic microfabrication defines a multiplexed sensing array; bottom-up fabrication of nanostructured microelectrodes then provides sensing elements. We analyzed a panel of mRNA samples for prostate cancer related gene fusions using the chip. We accurately identified gene fusions that correlate with aggressive prostate cancer and distinguished these from fusions associated with slower-progressing forms of the disease. The multiplexed nanostructured microelectrode integrated circuit reported herein provides direct, amplification-free, sample-to-answer in under 1 h using the 10 ng of mRNA readily available in biopsy samples.

16-Channel CMOS Impedance Spectroscopy DNA Analyzer With Dual-Slope Multiplying ADCs

We present a 16-channel, mixed-signal CMOS DNA analyzer that utilizes frequency response analysis (FRA) to extract the real and imaginary impedance components of the biosensor. Two computationally intensive operations, the multiplication and integration required by the FRA algorithm, are performed by an in-channel dual-slope multiplying ADC in the mixed-signal domain resulting in minimal area and power consumption. Multiplication of the input current by a digital coefficient is implemented by modulating the counter-controlled duration of the charging phase of the ADC. Integration is implemented by accumulating output digital bits in the ADC counter over multiple input samples. The 1.05 × 1.6 mm prototype fabricated in a 0.13 μm standard CMOS technology has been validated in prostate cancer DNA detection. Each channel occupies an area of only 0.06 mm2 and consumes 42 μW of power from a 1.2 V supply.

Nanostructured CMOS Wireless Ultra-Wideband Label-Free PCR-Free DNA Analysis SoC

A fully integrated 54-channel wireless fast-scan cyclic voltammetry DNA analysis SoC is presented. The microsystem includes 546 3D nanostructured and 54 2D gold DNA sensing microelectrodes as well as 54 pH sensors. Each channel consists of a chopper-stabilized current conveyer with dynamic element matching. It is utilized as the amperometric readout circuit with a linear resolution from 8.6 pA to 350 nA. The on-chip programmable waveform generator provides a wide range of user-controlled rate and amplitude parameters with a maximum scan range of 1.2 V, and scan rate ranging between 0.1 mV/sec to 300 V/sec. A digital ultra-wideband transmitter based on a delay line architecture provides wireless data communication with data rates of up to 50 Mb/sec while consuming 400 μW. The 3 mm × 3 mm prototype fabricated in a 0.13 μm standard CMOS technology has been validated in prostate cancer synthetic DNA detection with 10 aM label-free PCR-free detection limit. Each channel occupies an area of only 0.06 mm2 and consumes 42 μW of power from a 1.2 V supply.

Prototyping of wrinkled nano-/microstructured electrodes for electrochemical DNA detection.

Biosensing platforms are ideal for addressing the diagnostic needs of resource-poor areas; however, the translation of such systems from the laboratory to the point-of-need has been a slow process. Rapid prototyping methods that enable an application-specific biosensor to be created in a matter of hours from design to fabrication would expedite the clinical and field testing of such systems. Here, we demonstrate a benchtop method based on craft cutting and polymer-induced wrinkling for creating multiplexed electrochemical DNA biosensors. This fabrication method allows multiscale wrinkled electrodes with features in the millimeter to nanometer length scales to be created in a matter of hours. These wrinkled electrodes display an enhanced surface area compared to planar electrodes and are shown to be structurally tunable by changing the film thickness. We demonstrate that structural tunability of these electrodes is translatable to functional tunability as the density of surface-immobilized probe molecules can be manipulated using wrinkled electrodes of different thicknesses. Furthermore, a simple proof-of-concept electrocatalytic DNA biosensor is demonstrated for distinguishing between complementary and noncomplementary oligonucleotides.

Integrated nanostructures for direct detection of DNA at attomolar concentrations

We report an integrated chip that senses nucleic acid biomarkers at exceptionally low concentrations. To achieve such sensitivities we exploit four concepts. (1) Nanostructured electrodes allow efficient display of probe sequences. (2) The use of uncharged probe sequences lowers the background signal in our read-out system. (3) Electrocatalysis provides built-in amplification of the electrical signal that reports hybridization events. (4) An optimal self-assembled monolayer of thiol-functionalized probe molecules is best achieved with the aid of a short spacer molecule to confer enhanced accessibility. We show herein that via joint optimization along these four axes we achieve attomolar sensitivity.

Rapid prototyping of all-solution-processed multi-lengthscale electrodes using polymer-induced thin film wrinkling

Three-dimensional electrodes that are controllable over multiple lengthscales are very important for use in bioanalytical systems that integrate solid-phase devices with solution-phase samples. Here we present a fabrication method based on all-solution-processing and thin film wrinkling using smart polymers that is ideal for rapid prototyping of tunable three-dimensional electrodes and is extendable to large volume manufacturing. Although all-solution-processing is an attractive alternative to vapor-based techniques for low-cost manufacturing of electrodes, it often results in films suffering from low conductivity and poor substrate adhesion. These limitations are addressed here by using a smart polymer to create a conformal layer of overlapping wrinkles on the substrate to shorten the current path and embed the conductor onto the polymer layer. The structural evolution of these wrinkled electrodes, deposited by electroless deposition onto a nanoparticle seed layer, is studied at varying deposition times to understand its effects on structural parameters such as porosity, wrinkle wavelength and height. Furthermore, the effect of structural parameters on functional properties such as electro-active surface area and surface-enhanced Raman scattering is investigated. It is found that wrinkling of electroless-deposited thin films can be used to reduce sheet resistance, increase surface area, and enhance the surface-enhanced Raman scattering signal.