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This work initially describes the design of low-cost, naked-eye quantitative aptamer-based assays by using microfluidic paper-based analytical device (μPAD). Two new detection motifs are proposed for quantitative μPAD measurement without using external electronic readers, which depend on the length of colored region in a strip-like μPAD and the number of colorless detection microzones in a multi-zone μPAD. The length measuring method is based on selective color change of paper from colorless to blue-black via formation of iodine-starch complex. The counting method is conducted on the basis of oxidation-reduction reaction between hydrogen peroxide and potassium permanganate. Their utility is well demonstrated with sensitive, specific detection of adenosine as a model analyte with the naked eye in buffer samples and undiluted human serum. These equipment-free quantitative methods proposed thus hold great potential for the development of more aptamer-based assays that are simple, cost-efficient, portable, and user-friendly for various point-of-care applications particularly in resource-constrained environments.

A versatile point-of-care assay platform was developed for simultaneous detection of multiple targets based on a microfluidic paper-based analytic device (μPAD) using a target-responsive hydrogel to mediate fluidic flow and signal readout. An aptamer-cross-linked hydrogel was used as a target-responsive flow regulator in the μPAD. In the absence of a target, the hydrogel is formed in the flow channel, stopping the flow in the μPAD and preventing the colored indicator from traveling to the final observation spot, thus yielding a "signal off" readout. In contrast, in the presence of a target, no hydrogel is formed because of the preferential interaction of target and aptamer. This allows free fluidic flow in the μPAD, carrying the indicator to the observation spot and producing a "signal on" readout. The device is inexpensive to fabricate, easy to use, and disposable after detection. Testing results can be obtained within 6 min by the naked eye via a simple loading operation without the need for any auxiliary equipment. Multiple targets, including cocaine, adenosine, and Pb(2+), can be detected simultaneously, even in complex biological matrices such as urine. The reported method offers simple, low cost, rapid, user-friendly, point-of-care testing, which will be useful in many applications.

In this article, we describe a facile method named as Rubik's Cube stamping (RCS) for equipment-free fabrication of microfluidic paper-based analytical devices (μPADs). RCS is inspired by the worldwide ubiquitous RC toy and requires no specialized electric equipment other than a classical six-faced RC that is assembled with home-made small iron components. It can pattern various rosin microstructures in paper simply by either using different functional faces of the modified RC or applying its internal pivot mechanism to adjust the components' patterning forms on one functional face. Such a versatile stamping method is quite simple and inexpensive, and thus holds potential for producing rosin-patterned μPADs by untrained users in resource-limited environments such as small laboratories and private clinics, or even at home and in the field. Moreover, a set of one-channel devices are fabricated to design a point-of-care aptamer-based assay with near sample-in-answer-out capability that integrates enzymatic reactions for robust yet efficient signal amplification and a personal glucometer for portable, user-friendly, rapid and quantitative readout. Its utility is well demonstrated with the sensitive and specific detection of adenosine as a model target in buffer samples and undiluted human urine within several minutes. With the advantages of low cost, simplicity, portability, rapidity, and aptamer variety, this general point-of-care assay system reported here may find broad applications including home healthcare, field-based environmental monitoring or food analysis and emergency situations.

Here we report a self-powered origami paper analytical device (oPAD) that uses an aptamer to recognize an analyte, a glucose oxidase (GOx) tag to modify the relative concentrations of an electroactive redox couple, and a digital multimeter (DMM) to transduce the result of the assay. The sensor is self-powered in that it self-generates an electrical signal so that the read-out process is similar to testing a battery. The principle of the sensor is illustrated in Scheme 1. Briefly, the device is printed on a single piece of paper, folded into a three-dimensional (3D) configuration, and then laminated in plastic. An aliquot of sample is loaded at the inlet, split into two channels, and then directed to microbeads entrapped within the channels. In one channel, an aptamer immobilized on microbeads binds to the target and releases a GOx-labeled DNA strand that flows downstream. No aptamer is present on the microbeads in the other channel, which is used as a control. The split fluids terminate in an hour-glass-shaped, two-compartment electrochemical cell. The waist of the hour glass serves as a salt bridge between the two half cells. In one of the half cells, GOx catalyzes the oxidation of glucose, which in turn results in conversion of Fe(CN)63− to Fe(CN)64−.The difference in concentrations of Fe(CN)63− and Fe(CN)64− in the sensing half cell and control half cell results in a voltage that is used to charge a capacitor. When the switch (lower part of Scheme 1) is closed, the capacitor discharges through the DMM. The capacitor provides a high instantaneous current, in effect an amplified current, and hence a higher sensitivity than a direct current measurement.

The majority of bioassays utilize thermosensitive reagents (e.g., biomolecules) and laboratory conditions for analysis. The developing world, however, requires inexpensive, simple-to-perform tests that do not require refrigeration or access to highly trained technicians. To address this need, paper-based bioassays using gold nanoparticle (AuNP) colorimetric probes have been developed. In the two prototype DNase I and adenosine-sensing assays, blue (or black)-colored DNA-cross-linked AuNP aggregates were spotted on paper substrates. The addition of target DNase I (or adenosine) solution dissociated the gold aggregates into dispersed AuNPs, which generated an intense red color on paper within one minute. Both hydrophobic and (poly(vinyl alcohol)-coated) hydrophilic paper substrates were suitable for this biosensing platform; by contrast, uncoated hydrophilic paper caused "bleeding" and premature cessation of the assay due to surface drying. The assays are surprisingly thermally stable. During preparation, AuNP aggregate-coated papers can be dried at elevated temperatures (e.g., 90 degrees C) without significant loss of biosensing performance, which suggests the paper substrate protects AuNP aggregate probes from external nonspecific stimuli (e.g., heat). Moreover, the dried AuNP aggregate-coated papers can be stored for at least several weeks without loss of the biosensing function. The combination of paper substrates and AuNP colorimetric probes makes the final products inexpensive, low-volume, portable, disposable, and easy-to-use. We believe this simple, practical bioassay platform will be of interest for use in areas such as disease diagnostics, pathogen detection, and quality monitoring of food and water.

1. 1. The metabolism of [8-14C]adenosine was examined in human erythrocytes and erythrocyte lysates. 2. 2. The metabolism of [8-14C]adenosine in erythrocytes is largely dependent upon its concentration. 3. 3. Adenosine deaminase and adenosine kinase activities were assayed in crude erythrocyte lysates. No adenosine phosphorylase activity was detected. 4. 4. Adenosine deaminase exhibited a Km for adenosine of 4 · 10−5 M. Substrate inhibition of the enzyme was exhibited at concentrations of adenosine greater than 2 · 10−4 M. No inhibition of deaminase activity by equimolar or greater concentration of AMP, ADP, ATP, GDP, GTP, Pi, guanylic acid or 2,3-diphosphoglycerate was noted, but 20 % inhibition by high concentrations of adenine and inosine was observed. 5. 5. Adenosine kinase required ATP (Km = 4 · 10−4 M), adenosine (Km = 1.9 · 10−6 M), and Mg2+ for activity. No substrate inhibition by adenosine occurred at concentrations 20 times its Km. Adenosine 5′-triphosphate and Mg2+ were inhibitory at concentrations greater than 1.25 mM and 0.50 mM, respectively. The reaction of ATP with adenosine kinase was competitively inhibited by AMP, ADP, guanylic acid, GDP, IMP, and adenine. Adenosine 5′-triphosphate was variably replaceable as the phosphate donor by a wide range of triphosphates.

A method has been developed for the enzymic production of adenosine 5′-phosphosulfate labeled with either 14 C or 35 S. The procedure involves incubating AMP, Na 2 SO 3 , and K 3 Fe(CN) 6 with APS reductase from Thiobacillus denitrificans . Radioactivity is incorporated from either AMP- 14 C or Na 2 35 SO 3 . The product is isolated by column chromatography on DEAE-cellulose. Yields based on AMP converted are above 60%.