Detection Of Molecules In Solution Research Articles

(Invited) Carbon Nanotube Field Effect Transistor for Biological Detection in Liquids

Carbon nanotube (CNT) network films are an effective semiconducting platform for field effect transistor (FET) device applications. Their desirable properties include simple reproducible fabrication procedures1 with performances that are maintained during measurements in aqueous environments.2It is also well known that CNTFETs often show strong electronic responses to changes in their surrounding environment. Due to this one of the key challenges with electronic sensing is the reliable detection of a specific target. The emergence of synthetic receptors such as DNA aptamers which bind to targets with high selectivity3 has stimulated new opportunities for CNTFET biosensors.4 These short strands of DNA can be generated via a synthetic evolution process to bind with specificity for different types of targets including cells, proteins, viruses and small molecules.5 Once the correct sequence for a given target is discovered, DNA aptamers can be synthesized and readily functionalised for integration in biosensor devices. Figure 1 shows a schematic of a CNTFET biosensor, where the CNTs are directly functionalized with aptamers, and the sensor is operated in a liquid buffer gated through an Ag/AgCl electrode. The source and drain electrodes are encapsulated with an insulating material to ensure no leakage paths directly form the gate to the source, and the ensure all sensing is taking place on the CNTFET channel region. Although these CNTFET apatasensors have shown immense promise, some challenges remain. Is it possible to detect small molecules? What are the limiting factors of the CNTFET aptasensor platform? Each analyte and aptamer system presents different challenges with implications in the design of aptamers for operational sensors. For example, we have demonstrated a small molecule, 17 β-estradiol (E2),6 sensor based on CNTFET aptasensors using both 35-mer, 75-mer E2 aptamer sequences and a random 35-mer aptamer sequence.2 The 35-mer E2 aptamer functionalized CNTFETs show a clear increase in current whereas the 75-mer E2 aptamer functionalised CNTFET where the aptamer/E2 binding occurs beyond the Debye length, shows no obvious evidence of sensing. The performance of this and other CNTFET aptasensors will be discussed with emphasis on the role of electrostatic gating effects and the implications for future aptamer design. 1. Rouhi, N., Jain, D. & Burke, P. J. High-performance semiconducting nanotube inks: progress and prospects. ACS Nano 5, 8471–87 (2011). 2. Zheng, H. Y., A. Alsager, O., S. Wood, C., M. Hodgkiss, J. & O. V. Plank, N. Carbon nanotube field effect transistor aptasensors for estrogen detection in liquids. J. Vac. Sci. Technol. B 33, 06F904 (2015). 3. Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510 (1990). 4. So, H.-M. et al. Single-walled carbon nanotube biosensors using aptamers as molecular recognition elements. J. Am. Chem. Soc. 127, 11906–7 (2005). 5. Lee, J.-O. et al. Aptamers as molecular recognition elements for electrical nanobiosensors. Anal. Bioanal. Chem. 390, 1023–32 (2008). 6. Alsager, O. a, Kumar, S., Willmott, G. R., McNatty, K. P. & Hodgkiss, J. M. Small molecule detection in solution via the size contraction response of aptamer functionalized nanoparticles. Biosens. Bioelectron. 57, 262–8 (2014). Figure 1

Small molecule detection in solution via the size contraction response of aptamer functionalized nanoparticles

We demonstrate a simple new sensor design that exploits aptamer functionalized nanoparticles (NPs) to transduce the signal of aptamer receptors binding to target small molecules. An aptamer capable of binding to our target 17β-estradiol (E2) was isolated by SELEX with dissociation constant of 50nM and tethered to the surface of carboxylated polystyrene NPs. Upon exposing the aptamer functionalized NPs to E2 in buffered water, we use dynamic light scattering (DLS) and resistive pulse sensing (TRPS) to observe a distinct reduction of the conjugated particle size and a less negative zeta potential, which can be correlated to the E2 concentration in the lower nanomolar range. The sensor showed similar affinity towards other hormones of the E2 steroidal family and excellent discrimination against potential non-steroidal interfering agents. The simplicity of the sensing scheme makes it readily applicable to other low molecular weight targets, as we further demonstrate using a known adenosine aptamer. In addition to sensing, our method shows potential to guide the synthetic evolution of aptamers with better binding affinity and specificity.

A Low Noise Ion Channel Amplifier in a USB Pen Drive

Ion channels are transmembrane proteins responsible of ion exchange between intra- and extra-cellular environments. Ion channel play an essential role in many biological processes and their malfunctions are involved in several diseases or severe pathologies. As a result, ion channels are targets of numerous pharmaceutical compounds.The capability of ion channels to respond to different chemical-physical stimuli has inspired the design of hybrid sensors, where they are adopted as sensing units. This approach has already been employed for the detection of molecules in solution and for DNA sequencing.Both for biology and electronics extents, it is crucial to characterize ion channel electrical behavior with high accuracy and precision. This is achieved using low-noise acquisition equipments able to amplify ion channel picoamps-range currents.Commercially amplifiers for ion channel recording are bulky instrumentations and are limited in the number of channel simultaneously acquired due to their discrete-architecture electronic limitations. To overcome this weaknesses a single-microchip (integrated circuitry-based) amplifiers have been presented. Unfortunately they suffer from shortcomings like system integration, data elaboration and digital interface, resulting in unsettled systems.We developed an innovative low-noise technology for ion channel acquisition microchips that allow us to shrink in few squared millimeters the acquisition system improving the performances usually obtained with bulky instruments.We present our new technology integrated into a microchip and embedded into a USB pen ready to use. The fully comprehensive system includes the amplifier, the data elaboration, the digital interface and the data link.Furthermore, our technology allows us to configure the system for single or multi-channel acquisition, addressing the needs of emerging microfluidic techniques. Moreover, it offers an extremely flexible input interface and a user programmable stimulus, allowing different setups for different low-noise acquisition needs in different applications.

Nanoparticle Free Synthesis of Fluorescent Gold Nanoclusters at Physiological Temperature

This is a copy of the slides presented at the meeting but not formally written up for the volume. Single-molecule manipulation and detection of biomolecules has significantly advanced our understanding of the molecular movement, dynamics, and biological function of proteins. Fluorescence microscopy currently serves as one of the primary noninvasive techniques for the sensitive detection of molecules in solution and on surfaces. However, the performance and sensitivity of this laser-induced fluorescence technique is strongly influenced by the fluorescent labels attached to DNA, proteins or cells. Organic dyes have most commonly been used as fluorescent biolabels; however, they quickly photobleach, limiting the time scale over which molecular events can be followed. Quantum dots show great promise for fluorescence measurements due to their improved photophysical properties, such as size-tunable narrow emissions, large Stokes shifts and minimal photobleaching. However, problems still exist for the use of quantum dots as biolabels, including: photoblinking, toxic synthetic approaches, surface passivation issues and relatively large physical sizes that are comparable to proteins. Here, we report the synthesis of a new class of fluorescent labels, gold nanoclusters, which consist of several gold atoms (<1 nm in size) with strong fluorescence emission. These small fluorescent gold nanoclusters are synthesized at physiological temperature using poly(amidoamine) dendrimer as a template. Small blue emissive gold nanoclusters were produced without the use of a reductant and without concurrent nanoparticle formation. Gold nanoclusters with green and red emissions were synthesized using a mild reductant, also without nanoparticle formation. The studies of pH-dependent stability suggest that these fluorescent nanoclusters are very stable in a pH range of 6 - 8. These new approaches produce gold nanoclusters with a much higher yield and eliminate the toxicity of the previously reported process, resulting in a biologically compliant approach. This work is the first known report of fluorescent gold nanoclusters via a green-chemistry approach and without the formation of gold nanoparticles. This work is funded by Los Alamos Laboratory Directed research and Development program.

Synthesis of Aucore–Agshelltype bimetallic nanoparticles for single molecule detection in solution by SERS method

This paper reports the evolution of a new class of core–shell type, that is, Aucore–Agshell bimetallic nanoparticles by seed mediated technique for surface enhanced Raman scattering (SERS) study. Here it is demonstrated how to control the thickness of Ag-shell with the variation of gold seed (∼15 nm) to Ag ion concentration which in turn control the particle size in the range from 50 to 100 nm with increase of shell thickness. For 50 nm core–shell particles the thickness of the shell was ∼17 nm, for 70 nm particles the thickness was ∼27 nm and for 100 nm the thickness was ∼42 nm. SERS study was performed on those particles using the analyte crystal violet (CV) to examine the impact of the size and field effects of the bimetallics on SERS spectra. A surprising finding is that a small particle as low as 50 nm have been found to be highly efficient for SERS, even it enables the detection of a selected dye molecule down to single molecular level. The sensitivity of the SERS detection limit has been improved further with an activating reagent like NaCl. The newly modeled bimetallic system establishes a relationship between the local electromagnetic (EM) field effect and chemical effect (CE) on the enhancement of SERS spectra, which provides further insight into the enhancement mechanism of SERS.

Book Review: Single Molecule Detection in Solution—Methods and Applications. Edited by Christof Zander, Jörg Enderlein, and Richard A. Keller

Book Review: Single Molecule Detection in Solution—Methods and Applications. Edited by Christof Zander, Jörg Enderlein, and Richard A. Keller

Steps towards spatially resolved single molecule detection in solution

Spatially resolved single molecule detection in solution is a prerequisite for molecule tracking and provides new opportunities in single molecule manipulation, for example in a sample flow. A detector with fast timing properties and high spatial resolution is required for these purposes. We introduce a concept for spatially resolved optical single molecule detection in an epi-illuminated microscope using a novel kind of detector and a new algorithm in configurable hardware for intelligent data processing. The analysis is performed on a parallel hardware interface and includes burst detection. It can be extended to on-line spatial and temporal data analysis. This detector will be used in combination with a molecular sorter where molecules are sorted in a microstructured flow device made of silicon. In a first step towards such sorting in these microstructures, we show that a reliable detection of single molecules in silicon microstructures is possible in zero-dimensional detection volumes. The noise structure of the data is analysed in terms of Poissonian statistics and it is shown that in the smallest structure used (depth 20 μm) a signal-to-noise ratio of 40 is achieved.

Real-time observation of single molecules by confocal fluorescence microscopy

The ability to detect, identify, and manipulate individual molecules offer exciting possibilities in many fields, including chemical analysis, materials research, and the biological sciences. A particularly powerful approach is to combine the exquisite sensitivity of laser-induced fluorescence and the spatial localization and imaging capabilities of diffraction-limited or near-field optical microscopes. Unlike scanning tunneling microscopy (STM) and atomic force microscopy (AFM), which lack molecular specificity, optical spectroscopy and microscopy techniques can be used for real-time monitoring and molecular identification at nanometer dimensions or in ultrasmall volumes.We report the use of confocal fluorescence microscopy coupled with a diffraction-limit laser beam and a high-efficiency photodiode for real-time detection of single fluorescent molecules in solution at room temperature. Rigler and Eigen have also demonstrated single-molecule detection with a confocal microscope and fluorescence correlation spectroscopy. The probe (or sampling) volume is effectively an elongated cylinder, with its radius being determined by optical diffraction and length by spherical aberration.