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The determination of pH values is essential in many chemical, medical, and environmental monitoring processes, which has been relying on conventional pH meters (glass electrodes) for quantitation and pH test strips for qualitative (or semi-quantitative) assessment. In this work, we demonstrate a smartphone-based pH determination technique, which performs digital image analysis of commercial test strips, particularly the determination of either the dominant wavelength (λd) or complementary wavelength (λc) of the color image. In conjunction with a 3D-printed optical accessory (with a surface light source and a macro lens), the quality of captured images have been warranted, and the quantitation accuracy of 0.05 pH units has been achieved. More importantly, the performance of this smartphone-based pH reading system (namely "Smart-pH-Reader") was validated using multiple real-world samples, as the results are consistent with those determined with a standard pH meter. The Smart-pH-Reader is envisioned to be a simple, portable, and accurate tool for pH determination in the fields of environmental monitoring, medical diagnosis, and beyond.

In terms of how the signal varies in response to increased concentration of an analyte, sensors can be classified as either "signal-on" or "signal-off" format. While both types hold potentials to be sensitive, selective, and reusable, in many situations "signal-on" sensors are preferred for their low background signal and better selectivity. In this study, with the detection of lysozyme using its DNA aptamer as a trial system, for the first time we demonstrated that such an aptamer-based electrochemical biosensor can be converted from intrinsically "signal-off" to "signal-on" with the aid of a DNA exonuclease. The fact that the stepwise cleavage of antilysozyme aptamer catalyzed by Exonuclease I (Exo I) is entirely inhibited upon binding lysozyme leads to the selective removal of unbound DNA probes (thiolate anti-lysozyme DNA aptamer strands immobilized on gold electrode) upon the introduction of Exo I to the sensor. With the aid of electrostatically bound redox cations ([Ru(NH3)6]3+), we were able to quantitate the number of aptamer strands that are bound with lysozymes via conventional cyclic voltammetry (CV) measurements. We demonstrated that Exo I-assisted signal-on conversion protocol not only improves the sensing performance (10 times better limit of detection) but also promises a versatile strategy for DNA-based biosensor design, i.e., it can be readily adapted to other aptamer-protein binding systems (thrombin, as another example).

The macrocyclic cucurbit[7]uril (CB[7]) host has exhibited great application potential as a pharmaceutical excipient due to its versatile abilities to modulate the chemical/physical properties of drug molecules (guests) and to control their in vivo delivery and release (upon complexation). The formation of stable CB[7]@drug complexes is the prerequisite for these promising applications; we report herein a general assay strategy to quantitate the complexation based on competitive binding with surface-immobilized redox guests in conjunction with conventional electrochemical techniques (e.g., cyclic voltammetry). Particularly, by incubating a mixture of CB[7] and a drug molecule with ferrocene (Fc)-terminated self-assembled monolayers (SAMs) on gold, the competitive host@guest binding between the CB[7]@drug complex formed in solution and the CB[7]@Fc complex formed on surface can be quantified with direct cyclic voltammetry measurements. On the basis of the known concentrations of CB[7]/drug and electrochemically determined surface densities of free/complexed Fc groups, the formation constant of CB[7]@drug complex can be determined. With several drug molecules as examples, we have demonstrated the capability of this method for quantitative studies of the formation of supramolecular excipient@drug complexes that are of interest in pharmaceutical and biomedical sciences. More importantly, this work promises a general assay strategy that allows electrochemical quantitation of a wide range of electro-inactive analytes based on the competitive supramolecular host@guest binding at redox-tagged molecular interfaces.

Mechanoelectronic DNA nanoswitches refer to designed oligonucleotide constructs that are composed of conduction-interrupted duplex stems functionally coupled to ligand recognition motifs; they have been shown to undergo remarkable conduction switching upon binding molecular ligands/analytes. Herein we report a divergent pair of such mechanoelectronic DNA switches, the "signal-on" 3'AA-1 switch and the "signal-off" NB-1 switch, both activated by and responded to mercury ions (Hg2+) at nM levels. We first investigated their charge transport efficiency at a biochemical level, by studying how distinct base sequence at the switches' central three-way junction and at the recognition motif (capable of forming T-Hg2+-T metallo-base pairs) influences their overall conductivity. Gel electrophoresis assays revealed that the presence of two unpaired adenines (AA) at the junction led to "signal-on" behavior with increasing Hg2+ concentration; divergently, absence of these adenines led to a "signal-off" behavior. Upon immobilization on gold electrodes, both DNA switches, with enhanced and inhibited conductivity, respectively, showed excellent sensitivity as well as selectivity toward Hg2+ and can be regenerated for multicycle applications. The high performance of these devices, as both nanoswitches and biosensors with robust and reproducible properties, highlights their potential as an outstanding new class of DNA mechanoelectronic components with built-in biosensing capabilities.

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.

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.

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 interf...

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 quantit...

A sensitive and cost-effective method for the simultaneous quantitation of trace amounts of Hg(2+) and Pb(2+) in real-world samples has been developed using DNA molecular beacon probes bound to bona fide digital video discs (DVDs). With specially designed T-rich or G-rich loop sequences, the detection is based on the strong T-Hg(2+)-T coordination chemistry of Hg(2+) and the formation of G-quadruplexes induced by Pb(2+), respectively. In particular, the presence of metal cations leads to hairpin opening and exposure of the terminal biotin moiety for binding nanogold-streptavidin conjugates. The recognition signal was subsequently enhanced by gold nanoparticle-promoted silver deposition, which leads to quantifiable digital signals upon reading with a standard computer drive. This method exhibits a wide response range and low detection limits for both Hg(2+) and Pb(2+). In addition, the quantitative determination of heavy metals in food products (e.g., rice samples) has been demonstrated and the method compares favorably with other optical sensors developed recently.

On-site oral fluid testing for drugs of abuse has become prominent in order to take immediate administrative action in an enforcement process. Herein, we report a DVD technology-based indirect competitive immunoassay platform for the quantitative detection of drugs of abuse. A microfluidic approach was adapted to prepare multiplex immunoassays on a standard DVD-R, an unmodified multimode DVD/Blu-Ray drive to read signal, and a free disc-quality analysis software program to process the data. The DVD assay platform was successfully demonstrated for the simultaneous, quantitative detection of drug candidates (morphine and cocaine) in oral fluids with high selectivity. The detection limit achieved was as low as 1.0 ppb for morphine and 5.0 ppb for cocaine, comparable with that of standard mass spectrometry and ELISA methods.

Herein we report a combined electrochemical and ESI-MS study of the enzymatic hydrolysis efficiency of DNA self-assembled monolayers (SAMs) on gold, platform systems for understanding nucleic acid surface chemistry and for constructing DNA-based biosensors. Our electrochemical approach is based on the comparison of the amounts of surface-tethered DNA nucleotides before and after Exonuclease I (Exo I) incubation using electrostatically bound [Ru(NH3)6]3+ as redox indicators. It is surprising to reveal that the hydrolysis efficiency of ssDNA SAMs does not depend on the packing density and base sequence, and that the cleavage ends with surface-bound shorter strands (9-13 mers). The ex-situ ESI-MS observations confirmed that the hydrolysis products for ssDNA SAMs (from 24 to 56 mers) are dominated with 10-15 mer fragments, in contrast to the complete digestion in solution. Such surface-restrained hydrolysis behavior is due to the steric hindrance of the underneath electrode to the Exo I/DNA binding, which is essential for the occurrence of Exo I-catalyzed processive cleavage. More importantly, we have shown that the hydrolysis efficiency of ssDNA SAMs can be remarkably improved by adopting long alkyl linkers (locating DNA strands further away from the substrates).

Compact disc-based bioassays have been developed as novel point-of-care (POC) tools for various applications in chemical analysis and biomedical diagnosis. For the fabrication of assay discs, the surface patterning and sample introduction have been restricted to manual delivery that is unfavorable for on-demand high throughput medical screening. Herein, we have adapted a conventional inkjet printer to prepare bioassays on regular DVD-Rs and accomplished quantitative analysis with a multimode DVD/Blu-Ray optical drive in conjunction with free disc diagnostic software. The feasibility and accuracy of this method have been demonstrated by the quantitative analysis of inkjet-printed biotin-streptavidin binding assays on DVD, which serves as a trial system for other complex, medically relevant sandwich-format or competitive immunoassays.

A method for the convenient detection of lead at the parts-per-billion (ppb)-level has been developed; it uses a conventional compact disc (CD) as the platform for preparing DNAzyme assays and an unmodified optical drive of ordinary desktop/laptop computers as the readout device. In particular, by immobilization of Pb(2+) -specific DNAzyme sensing constructs on the "transparent side" of a conventional CD-R via mild surface reactions, the Pb(2+) concentration can be determined by a free diagnostic program that checks the error distribution on the CD (i.e., it extracts the number of errors in a prerecorded audio file). The reading errors increase monotonically over a wide range of Pb(2+) concentrations (from 10 nM to 1 mM), and the selectivity is confirmed by testing several other divalent cations (Zn(2+), Ba(2+), Mg(2+), Ca(2+), Cu(2+), and Hg(2+))

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.

The ion-exchange kinetics of metal cation binding to and dissociation from thiolate-DNA monolayers on gold can be monitored by a simple electrochemical protocol. The apparent first-order rate constants were obtained by analyzing the time-dependent voltammetric behavior of the redox cation [Ru(NH3)6]3+. It was found that the binding kinetics is dominated by the structural nature of the film; i.e., the apparent first-order rate constant (kapp) decreases significantly upon increasing the surface density of DNA strands. Dissociation rate constants were obtained by transferring the incubated electrode into redox-free buffer solution. The kinetic data augment our fundamental understanding of metal ion-DNA interactions and are critical to ensure the accuracy and reliability of experimental DNA detection protocols.

To examine DNA-modified surfaces, we have developed a simple, convenient, and reliable procedure based on the voltammetric response of multiply charged transition metal cations (such as [Ru(NH3)6]3+) bound electrostatically to the DNA probes. At micromolar concentrations of the redox molecules in the electrolyte, the reduction and oxidation waves resulting from the immobilized cations on DNA-modified electrodes are well defined, stable, and reproducible. The surface densities of both single- and double-stranded oligonucleotides were accurately determined by integration of the peak for reduction of [Ru(NH3)6]3+ to [Ru(NH3)6]2+. In addition, the binding constant and electron-transfer rate constant of [Ru(NH3)6]3+ on DNA-modified electrodes were evaluated with the help of classical models. The present research provides not only an applicable and simple protocol for the quantitation of DNA probes on chips but also a versatile and powerful tool for the investigation of the binding activity and electron-transfer kinetics of cationic analytes on DNA-modified surfaces.

In this paper, we describe a practical method of using gold films constructed from recordable compact disks (CD-Rs) as simple, inexpensive, and micropatterned conductive substrates for the fabrication of inorganic material microstructures. Extending from their application for the fabrication of self-assembled monolayers (SAMs) reported recently, bare and SAM-modified CD-R gold substrates have been used for template-directed electrodeposition of zirconia (ZrO2) thin films (i.e., the controlled formation of zirconia thin films on the different areas of the prefabricated, micrometer mountain-valley CD-R gold substrate surfaces). The present results demonstrate that the variation of the functional groups of the selected SAMs combined with electrodynamic control can be very successful to "customize" the formation and microstructure of functional inorganic thin films, which hold promise for modern technological applications.

Gold substrates for self-assembly were constructed from recordable compact disks (CD-Rs) with simple and straight-forward wet-chemical treatment. In particular, they were made into desired sizes and shapes that satisfy different experimental needs. Self-assembled monolayers (SAMs) of long-chain alkanethiols (e.g., n-C18H37SH) adsorbed on gold substrates prepared from CD-Rs have been characterized by wetting measurements, electrochemistry, infrared (IR) spectroscopy, and scanning tunneling microscopy (STM). The results showed that there were no distinct differences in the quality and structure between these monolayers and SAMs formed on standard gold substrates (i.e., prepared from vacuum evaporation of pure gold onto glass slides). The present work demonstrates the applicability of recordable CDs as inexpensive, simple, and versatile gold substrates for self-assembly studies.

Determination of the surface pKa of self-assembled monolayers (SAMs) at aqueous buffer/gold interfaces using surface-enhanced Raman scattering (SERS) measurements, namely, SERS titration, is reported for the first time. From the analysis of pH-dependent SERS spectra of 4-mercaptopyridine monolayers on gold, the pK1/2 (3.9 ± 0.2) was determined. By further correlating the surface pH value with the bulk pH value in terms of the Gouy−Chapmann model, the surface pKa (5.3 ± 0.3) was evaluated accordingly. Compared to the conventional contact angle titration, SERS titration has the advantage of giving further insight into the microscopic and dynamic information of the surface-confined functional groups. In addition, such a technique has high sensitivity, micrometer spatial resolution, and chemical selectivity.

Mucin 1 (MUC1) is a glycoprotein expressed on most epithelial cell surfaces, which has been confirmed as a useful biomarker for the diagnosis of early cancers. In this paper, we report an aptamer-based, quantitative detection protocol for MUC1 using a 3-component DNA hybridization system with quantum dot (QD)-labeling: in the absence of MUC1 peptides, strong fluorescence is observed upon mixing the three specially designed DNA strands (quencher, QD-labeled reporter, and the MUC1 aptamer stem); in the presence of MUC1 peptides, a successive decrease in fluorescence intensity is detected since the MUC1 peptide binds to the aptamer strand in such a way to allow the quencher and fluorescence reporter to be brought into close proximity (which leads to the occurrence of fluorescence resonance energy transfer, FRET, between the quencher and QD). The detection limit for MUC1 with this novel approach is in the nanomolar (nM) level, and a linear response can be established for the approximate range found in blood serum. This study also provided further insight into the aptamer/analyte binding site/mode for MUC1, which augments the possibility of improving this detection methodology for the early diagnosis of different types of epithelial cancers of large populations.

We report herein a digital signal readout protocol for screening disk-based bioassays with standard optical drives of ordinary desktop/notebook computers. Three different types of biochemical recognition reactions (biotin-streptavidin binding, DNA hybridization, and protein-protein interaction) were performed directly on a compact disk in a line array format with the help of microfluidic channel plates. Being well-correlated with the optical darkness of the binding sites (after signal enhancement by gold nanoparticle-promoted autometallography), the reading error levels of prerecorded audio files can serve as a quantitative measure of biochemical interaction. This novel readout protocol is about 1 order of magnitude more sensitive than fluorescence labeling/scanning and has the capability of examining multiplex microassays on the same disk. Because no modification to either hardware or software is needed, it promises a platform technology for rapid, low-cost, and high-throughput point-of-care biomedical diagnostics.

This paper reports a simple electrochemical approach for the detection of the ubiquitous protein lysozyme using aptamer-modified electrodes. Anti-lysozyme DNA aptamers were immobilized on gold surfaces by means of self-assembly, for which the surface density of aptamers was determined by cyclic voltammetric (CV) studies of redox cations (e.g., [Ru(NH3)6]3+) bound to the surface via electrostatic interaction with the DNA phosphate backbone. Upon incubation of the electrode with a solution containing lysozyme, the CV response of surface-bound [Ru(NH3)6]3+ changed substantially, and the relative decrease in the integrated charge of the reduction peak can be tabulated as a quantitative measure of the protein concentration. It is significant that the on-chip protein/aptamer binding constant and the optimized surface density to achieve the best detection limit can be evaluated. This biosensor is label-free and offers an alternative, sensitive, and versatile method for protein detection, which is beneficial to the ever-growing interests of fabricating portable bioanalytical devices with simple electrical readout protocols.

A mild and efficient surface activation protocol to convert polycarbonate (PC) substrates, e.g., plastic bases of compact disks, to biochip platforms for DNA probe immobilization and target detection is described. The preparation procedure (activation, patterning, and coupling) is simple and effective; the on-chip hybridization is sensitive and selective. Particularly, UV/ozone treatment of PC sheets produces a hydrophilic surface with a high density of reactive carboxylic acid groups [(4.8 +/- 0.2) x 10-10 mol/cm2] in less than 10 min at ambient conditions, and no significant aging or physical damage to the substrate is observed. Covalent immobilization of DNA probes via both passive (reagent-less photopatterning and coupling in bulk solution phase) and flow-through (creation of microarrays with microfluidic channel plates) procedures has been demonstrated. Subsequent hybridization shows uniform and strong fluorescent signals for complementary target DNA and allows clear discrimination between fully complementary targets and strands with a single base-pair mismatch. The surface chemistry described herein will facilitate the development of disposable plastic biochips (not limited to DNA microarrays) and the fabrication of biomedical devices that are readable with conventional optical drives.