Binary Thiolate DNA/Ferrocenyl Self-Assembled Monolayers on Gold: A Versatile Platform for Probing Biosensing Interfaces

The properties of DNA self-assembled monolayers (SAMs) have strong influences on the interfacial DNA–analyte binding behavior, which further affect the performance of biosensors built upon. In this work, we prepared binary thiolate DNA/6-ferrocenyl-1-hexanethiol (FcC6SH) SAMs on gold (DNA/FcC6S-Au) for convenient electrochemical characterization and subsequent data analysis. Our cyclic voltammetric (CV) studies confirmed that the redox responses of surface-tethered Fc and electrostatically bound [Ru­(NH₃)₆]³⁺ are capable of providing quantitative information regarding the DNA film properties, including the surface density, structural heterogeneity, and molecular orientation under different preparation and measurement conditions. With the binary thiolate DNA/FcC6S-Au SAM prepared in the conventional post-assembly exchange protocol as a trial system, we are demonstrating the capability of introducing redox-active thiols as passivating and labeling reagents for preparing many other DNA-based biosensing interfaces via varied assembly steps and under different measurement conditions

A Mechano-Electronic DNA Switch

We report a new kind of DNA nanomachine that, fueled by Hg²⁺ binding and sequestration, couples mechanical motion to the multiply reversible switching of through-DNA charge transport. This mechano-electronic DNA switch consists of a three-way helical junction, one arm of which is a T-T mismatch containing Hg²⁺-binding domain. We demonstrate, using chemical footprinting and by monitoring charge-flow-dependent guanine oxidation, that the formation of T-Hg²⁺-T base pairs in the Hg²⁺-binding domain sharply increases electron–hole transport between the other two Watson–Crick-paired stems, across the three-way junction. FRET measurements are then used to demonstrate that Hg²⁺ binding/dissociation, and the concomitant increase/decrease of hole transport efficiency, are strongly linked to specific mechanical movements of the two conductive helical stems. The increase in hole transport efficiency upon Hg²⁺ binding is tightly coupled to the movement of the conductive stems from a bent arrangement toward a more linear one, in which coaxial stacking is facilitated. This switch offers a paradigm wherein the performance of purely mechanical work by a nanodevice can be conveniently monitored by electronic measurement

Metastable Molecular Metal–Semiconductor Junctions

We demonstrate herein how to mechanically modulate the electrical properties of metastable molecular junctions, i.e., mercury–silicon junctions modified with “mobile” octadecanethiolate (C18) self-assembled monolayers (SAMs). By enlarging the mercury drop contact or changing its shape, the current density–voltage response of these molecular junctions vary remarkably from rectifying (off) to ohmic (on). More importantly, such switching behavior is reversible and reproducible when the shape of the mercury drop is changed from spherical to elliptical and vice versa (by pressing and releasing the mercury drop). Evaluation of the rectification ratio and effective barrier height of these molecular junctions enables determination of the threshold surface area of the mercury contact for the modulated electrical switching

Adenosine-Triggered Elimination of Methylene Blue Noncovalently Bound to Immobilized Functional dsDNA-Aptamer Constructs

Immobilization and electrochemical characterization of specially designed functional DNA–aptamer constructs are of great importance for the development of versatile biosensors (not limited to gene analysis) and the investigation of molecular interactions between DNA and other molecules. We have constructed a “DNA conformational switch” by incorporating the antiadenosine aptamer sequence in the middle of an otherwise cDNA double helix, as its structural change responds to the presence of small molecule ligands (e.g., adenosine). In particular, methylene blue (MB) was used as a model system to probe the rather complex interaction modes between small redox molecules and the dsDNA–aptamer construct. Besides intercalating with the double-stranded DNA stem, MB can stack with a single guanine base in the relatively unstructured aptamer domain or electrostatically bind to the DNA backbone. The decreased surface density of MB after adenosine binding indicated that the ligand-gated structural change of the dsDNA–aptamer construct can eliminate MB molecules that were originally bound to the aptamer domain but not those in the complementary stem

Analyte-Driven Switching of DNA Charge Transport: <i>De Novo</i> Creation of Electronic Sensors for an Early Lung Cancer Biomarker

A general approach is described for the <i>de novo</i> design and construction of aptamer-based electrochemical biosensors, for potentially any analyte of interest (ranging from small ligands to biological macromolecules). As a demonstration of the approach, we report the rapid development of a made-to-order electronic sensor for a newly reported early biomarker for lung cancer (CTAP III/NAP2). The steps include the <i>in vitro</i> selection and characterization of DNA aptamer sequences, design and biochemical testing of wholly DNA sensor constructs, and translation to a functional electrode-bound sensor format. The working principle of this distinct class of electronic biosensors is the enhancement of DNA-mediated charge transport in response to analyte binding. We first verify such analyte-responsive charge transport switching in solution, using biochemical methods; successful sensor variants were then immobilized on gold electrodes. We show that using these sensor-modified electrodes, CTAP III/NAP2 can be detected with both high specificity and sensitivity (<i>K</i>_d ∼1 nM) through a direct electrochemical reading. To investigate the underlying basis of analyte binding-induced conductivity switching, we carried out Förster Resonance Energy Transfer (FRET) experiments. The FRET data establish that analyte binding-induced conductivity switching in these sensors results from very subtle structural/conformational changes, rather than large scale, global folding events. The implications of this finding are discussed with respect to possible charge transport switching mechanisms in electrode-bound sensors. Overall, the approach we describe here represents a unique design principle for aptamer-based electrochemical sensors; its application should enable rapid, on-demand access to a class of portable biosensors that offer robust, inexpensive, and operationally simplified alternatives to conventional antibody-based immunoassays

Host–Guest Interaction at Molecular Interfaces: Cucurbit[7]uril as a Sensitive Probe of Structural Heterogeneity in Ferrocenyl Self-Assembled Monolayers on Gold

Herein, we combine host–guest recognition chemistry and electrochemical analysis to demonstrate that the nanometer-size, supramolecular hosts can be adapted as sensitive probes for structural heterogeneity in organized molecular assemblies on a surface. In particular, we carried out thorough cyclic voltammetric (CV) studies to evaluate the binding of cucurbit[7]­uril on mixed ferrocenylundecanethiolate/n-alkanethiolate self-assembled monolayers (SAMs) on gold (FcC11S-/CmS-Au) prepared with different methods (coadsorption vs postassembly exchanges) and with varied diluting n-alkanethiols. On the basis of the distinct CV responses of CB[7]@Fc complex and free Fc on the SAM surfaces, we were able to determine the conversion ratio from Fc to CB[7]@Fc, a direct indication of its overall density and uniformity. We have shown that the FcC11S-/C8S-Au prepared by coadsorption in a binary solution with low mole fraction of FcC11SH (5%) and by exchanging preassembled C8S-Au SAM with FcC11SH for a short time (1 min) has the “ideal” structure with isolated and uniformly distributed Fc groups on the surface. In contrast, with similar Fc surface coverage, the FcC11S-/C8S-Au prepared by exchanging FcC11S-Au with C8SH for a prolonged time (20 h) has clustered and nonuniformly distributed Fc groups at the surface. While consistent with previous observations based on conventional electrochemical or microscopic studies, the present finding expands the capability of host–guest chemistry as a new tool to probe the structures of organized molecular assemblies at the nanometer scale

Mobile App-Based Quantitative Scanometric Analysis

The feasibility of using smartphones and other mobile devices as the detection platform for quantitative scanometric assays is demonstrated. The different scanning modes (color, grayscale, black/white) and grayscale converting protocols (average, weighted average/luminosity, and software specific) have been compared in determining the optical darkness ratio (ODR) values, a conventional quantitation measure for scanometric assays. A mobile app was developed to image and analyze scanometric assays, as demonstrated by paper-printed tests and a biotin-streptavidin assay on a plastic substrate. Primarily for ODR analysis, the app has been shown to perform as well as a traditional desktop scanner, augmenting that smartphones (and other mobile devices) promise to be a practical platform for accurate, quantitative chemical analysis and medical diagnostics

Integrated Smartphone-App-Chip System for On-Site Parts-Per-Billion-Level Colorimetric Quantitation of Aflatoxins

We demonstrate herein an integrated, smartphone-app-chip (SPAC) system for on-site quantitation of food toxins, as demonstrated with aflatoxin B1 (AFB1), at parts-per-billion (ppb) level in food products. The detection is based on an indirect competitive immunoassay fabricated on a transparent plastic chip with the assistance of a microfluidic channel plate. A 3D-printed optical accessory attached to a smartphone is adapted to align the assay chip and to provide uniform illumination for imaging, with which high-quality images of the assay chip are captured by the smartphone camera and directly processed using a custom-developed Android app. The performance of this smartphone-based detection system was tested using both spiked and moldy corn samples; consistent results with conventional enzyme-linked immunosorbent assay (ELISA) kits were obtained. The achieved detection limit (3 ± 1 ppb, equivalent to μg/kg) and dynamic response range (0.5–250 ppb) meet the requested testing standards set by authorities in China and North America. We envision that the integrated SPAC system promises to be a simple and accurate method of food toxin quantitation, bringing much benefit for rapid on-site screening

Exonuclease I‑Hydrolysis Assisted Electrochemical Quantitation of Surface-Immobilized DNA Hairpins and Improved HIV‑1 Gene Detection

The complete formation of stem-loop (i.e., hairpin) configuration on chip surface is of particular importance for the application of hairpin DNA (hpDNA) in building biosensors for various analytes with optimized performance. We report herein a convenient electrochemical protocol for evaluating the yield of hairpin DNA conformations upon self-assembly on electrode surface. As of the different hydrolysis capability of Exonuclease I (Exo I) toward single-stranded DNA (ssDNA) and hpDNA, we can selectively remove ssDNA from electrode but retain hpDNA strands; based on the changes in the cyclic voltammetric (CV) responses using [Ru­(NH₃)₆]³⁺ as redox indicators, we can then determine the fraction of hairpin configurations in mixed DNA self-assembled monolayers (SAMs). It was discovered that the molar fraction of hairpin configuration formed on the surface is considerably lower than that in the binary deposition solution (containing both ssDNA and hpDNA). The accuracy of the Exo I-assisted electrochemical quantitative protocol has been validated by standard DNA hybridization experiments; the relationship between the overall DNA packing density and the yield of hairpin configurations was also evaluated. More importantly, taking HIV-1 gene detection as a trial system, the hpDNA-based biosensor shows significantly improved detection limit and broadened response range upon the background reduction by Exo I-catalyzed hydrolysis

Blu-ray Technology-Based Quantitative Assays for Cardiac Markers: From Disc Activation to Multiplex Detection

Acute myocardial infarction (AMI) is the leading cause of mortality and morbidity globally. To reduce the number of mortalities, reliable and rapid point-of-care (POC) diagnosis of AMI is extremely critical. We herein present a Blu-ray technology-based assay platform for multiplex cardiac biomarker detection; not only off-the-shelf Blu-ray discs (BDs) were adapted as substrates to prepare standard immunoassays and DNA aptamer/antibody hybrid assays for the three key cardiac marker proteins (myoglobin, troponin I, and C-creative protein) but also an unmodified optical drive was directly employed to read the assay results digitally. In particular, we have shown that all three cardiac markers can be quantitated in their respective physiological ranges of interest, and the detection limits achieved are comparable with conventional enzyme-linked immunosorbent assay (ELISA) kits. The Blu-ray assay platform was further validated by measuring real-world samples and establishing a linear correlation with the simultaneously obtained ELISA data. Without the need to modify either the hardware (Blu-ray discs and optical drives) or the software driver, this assay-on-a-BD technique promises to be a low-cost user-friendly quantitative tool for on-site chemical analysis and POC medical diagnosis