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Biocompatible hydrogels for tissue regeneration/replacement and drug release with specific architectures can be obtained by three-dimensional bioprinting techniques. The preservation of the higher order structure of the proteins embedded in the hydrogels as drugs or modulators is critical for their biological activity. Solution nuclear magnetic resonance (NMR) experiments are currently used to investigate the higher order structure of biotherapeutics in comparability, similarity, and stability studies. However, the size of pores in the gel, protein–matrix interactions, and the size of the embedded proteins often prevent the use of this methodology. The recent advancements of solid-state NMR allow for the comparison of the higher order structure of the matrix-embedded and free isotopically enriched proteins, allowing for the evaluation of the functionality of the material in several steps of hydrogel development. Moreover, the structural information at atomic detail on the matrix–protein interactions paves the way for a structure-based design of these biomaterials.

Solid supported colorimetric sensing arrays have the advantage of portability and ease of use when deployed in the field, such as crime scenes, disaster zones, or in war zones, but many sensor arrays require complex fabrication methods. Here, we report a practical method for the fabrication of 4 × 4 colorimetric sensor arrays, which are printed on nylon membranes, using a commercially available inkjet printer. In order to test the efficacy of the printed arrays, they were exposed to 43 analytes at concentrations ranging from 0.001 to 3.0 M for a total of 559 samples of inorganic and organic acids or bases including hydrochloric, acetic, phthalic, malonic, picric, and trifluoroacetic acid, ammonium hydroxide, sodium hydroxide, lysine, and water as the control. Colorimetric data from the imaged arrays was analyzed with linear discriminant analysis and k-nearest neighbors to determine the analyte and concentration with ∼88-90% accuracy. Overall, the arrays have impressive analytical power to identify a variety of analytes at different concentrations while being simple to fabricate.

Early screening of clinically relevant pathogens in the environment is a highly desirable goal in clinical care, providing precious information that will improve patient-care outcomes. In this work, a glove-based electrochemical sensor has been designed for point-of-use screening of Pseudomonas aeruginosa’s virulence factors. The methodology used for the elaboration of the fabric platform relied on printing the conductive inks on the index and middle fingers of the glove, with the goal of screening pyocyanin and pyoverdine targets. The analytical signatures of the analytes were recorded in about 4 min, via the rapid and selective square-wave-voltammetry technique. Finger-based sensors display good performance and discrimination between the targets and potential interferences, along with good reproducibility. The sensors featured linearity over the 0.01–0.1 μM range for pyocyanin and 5–50 μM range for pyoverdine, with sensitivities of 2.51 μA/μM for pyocyanin and 1.09 nA/μM for pyoverdine (R2 = 0.990 and 0...

We report on the development of a novel and flexible online digital polymerase chain reaction (dPCR) system. The system was composed of three parts: an inkjet for generating the droplets, a coiled fused-silica capillary for thermal cycling, and a laser-induced fluorescence detector (LIFD) for positive droplet counting. Upon inkjet printing, monodisperse droplets were continuously generated in the oil phase and then introduced into the capillary in the form of a stable dispersion. The droplets containing one or zero molecules of target DNA passed through the helical capillary that was attached to a cylindrical thermal cycler for PCR amplification, resulting in the generation of fluorescence for the DNA-positive droplet. After 36 PCR cycles, the fluorescence signal intensity was detected by laser-induced fluorescence located at the downstream of the capillary, followed by a positive/negative counting. The present system was successfully applied to the absolute quantification of the HPV sequence in Caski cells with dynamic ranges spanning 4 orders of magnitude.

This study describes a method to investigate the separation of cells by capillary electrophoresis (CE) coupled with inkjet printing system. The results validated the feasibility of inkjet printing for mammalian cells to achieve the drop-on-demand and convenient sampling into capillary then zone electrophoresis was applied to separate different cells according to their electrophoretic mobility, finally the peak signal were measured by UV detector. Linear relationship between the peak area and the droplet number was obtained within the range of 25-400 drops (R2 = 0.996) at a fixed cell concentration 106/mL, indicating that this system could be used for rapid and accurate quantification of cells.

Equilibrium dialysis is a simple and effective technique used for investigating the binding of small molecules and ions to proteins. A three-dimensional (3D) printer was used to create a device capable of measuring binding constants between a protein and a small ion based on equilibrium dialysis. Specifically, the technology described here enables the user to customize an equilibrium dialysis device to fit their own experiments by choosing membranes of various material and molecular-weight cutoff values. The device has dimensions similar to that of a standard 96-well plate, thus being amenable to automated sample handlers and multichannel pipettes. The device consists of a printed base that hosts multiple windows containing a porous regenerated-cellulose membrane with a molecular-weight cutoff of ~3500 Da. A key step in the fabrication process is a print-pause-print approach for integrating membranes directly into the windows subsequently inserted into the base. The integrated membranes display no leaking upon placement into the base. After characterizing the system’s requirements for reaching equilibrium, the device was used to successfully measure an equilibrium dissociation constant for Zn2+ and human serum albumin (Kd = (5.62 ± 0.93) × 10−7 M) under physiological conditions that is statistically equal to the constants reported in the literature.

In this paper, we introduce a novel microfluidic combinatorial synthesis platform, referred to as Microfluidic Print-to-Synthesis (MPS), for custom high-throughput and automated synthesis of a large number of unique peptides in a microarray format. The MPS method utilizes standard Fmoc chemistry to link amino acids on a polyethylene glycol (PEG)-functionalized microdisc array. The resulting peptide microarrays permit rapid screening for interactions with molecular targets or live cells, with low nonspecific binding. Such combinatorial peptide microarrays can be reliably prepared at a spot size of 200 μm with 1 mm center-to-center distance, dimensions that require only minimal reagent consumption (less than 30 nL per spot per coupling reaction). The MPS platform has a scalable design for extended multiplexibility, allowing for 12 different building blocks and coupling reagents to be dispensed in one microfluidic cartridge in the current format, and could be further scaled up. As proof of concept for the MPS platform, we designed and constructed a focused tetrapeptide library featuring 2560 synthetic peptide sequences, capped at the N-terminus with 4-[( N'-2-methylphenyl)ureido]phenylacetic acid. We then used live human T lymphocyte Jurkat cells as a probe to screen the peptide microarrays for their interaction with α4β1 integrin overexpressed and activated on these cells. Unlike the one-bead-one-compound approach that requires subsequent decoding of positive beads, each spot in the MPS array is spatially addressable. Therefore, this platform is an ideal tool for rapid optimization of lead compounds found in nature or discovered from diverse combinatorial libraries, using either biochemical or cell-based assays.

Three-dimensional (3D) printing has undergone an exponential growth in popularity due to its revolutionary and near limitless manufacturing capabilities. Recent trends have seen this technology utilized across a variety of scientific disciplines, including the measurement sciences, but precise fabrication of optical components for high-performance biosensing has not yet been demonstrated. We report here 3D printing of high-quality, custom prisms by stereolithography that enable Kretschmann-configured plasmonic sensing of bacterial toxins. Simple benchtop polishing procedures render a smooth surface that supports propagation of surface plasmon polaritons with a deposited gold layer, which exhibit high bulk refractive index sensitivities and are capable of discriminating trace levels of cholera toxin on a supported lipid membrane interface. Further evidence of the flexibility of this manufacturing technique is demonstrated with printed prisms of varied geometries and in situ monitoring of nanoparticle growth by total internal reflection spectroscopy. This work represents the first example of 3D printed light-guiding sensing platforms and demonstrates the versatility and broad perspective of 3D printing in optical detection.

The first paper-based polyion-sensitive optodes are reported. Dinonylnaphthalene sulfonic acid (a cation exchanger) and chromoionophore I (a lipophilic optical pH indicator) are printed on filter paper in the absence of any plasticizer and/or additional hydrophobic polymeric phase. The resulting optodes exhibit sensitive colorimetric response to polycations such as protamine but not to small inorganic cations because only polycations are able to form cooperative ion pairs with dinonylnaphthalenesulfonate adsorbed to the cellulose paper. The color change of the optode is recorded via an iPhone camera and analyzed by an iPhone App. The protamine-sensing optode platform is used to indirectly detect protease activity (trypsin) based on proteolytic digestion of protamine, and polyanions (pentosan polysulfate and heparin) based on the strong binding reaction of polyanions with protamine. The indirect sensing system is further simplified on a multilayer membrane device that consists of an optode paper site modified with buffer to prevent optode dependence on sample pH, and an underlying cellulose acetate filter membrane coated with protamine to eliminate addition of the indicator polycation into the sample. The detection of pentosan polysulfate concentrations in an undiluted urine sample is successfully demonstrated via this approach. Lastly, it is shown that plasticizer-free polyanion-sensitive optodes based on an adsorbed layer of quaternary ammonium type anion exchanger and a phenolic azo type proton chromoionophore can also be fabricated directly on cellulose paper strips.

In this study, a paper-based point-of-care (POC) colorimetric biosensor was developed for the detection of lactate dehydrogenase in serum using a nonporous, oxygen impermeable reversibly gelling polysaccharide material based on pullulan. The pullulan could be printed onto paper surfaces along with all required assay reagents, providing a means for high-stability immobilization of all reagents on paper. Serum containing lactate dehydrogenase (LDH) was directly spotted on to the pullulan-coated bioactive paper and provided quantitative colorimetric data that was comparable to that obtained with a conventional plate-reader method. The paper strip was found to be highly stable and could be stored at 4 °C for at least 10 weeks with no loss in performance, as compared to a complete loss in performance within 1 day when the reagents were printed without the stabilizing polysaccharide. The ease of fabrication coupled with the high stability of the printed reagents provides a facile platform for easily manufactured POC sensors.

Detection of infectious viruses and disease biomarkers is of utmost importance in clinical screening for effective identification and treatment of diseases. We demonstrate here the use of liposome rupture impact voltammetry for the qualitative detection of model amphipathic viral peptide on a screen-printed electrode. This novel, proof-of-concept method was proposed for the quick and reliable detection of viruses by nonfaradaic liposome rupture impact voltammetry with the aid of 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes. This provides an avenue for the development of future on-site, point-of-care detection devices for medical and biological applications.

The development of transparency-based electrochemical and paper-based colorimetric analytic detection platforms is presented as complementary methods for food and waterborne bacteria detection from a single assay. Escherichia coli and Enterococcus species, both indicators of fecal contamination, were detected using substrates specific to enzymes produced by each species. β-galactosidase (β-gal) and β-glucuronidase (β-glucur) are both produced by E. coli, while β-glucosidase (β-gluco) is produced by Enterococcus spp. Substrates used produced either p-nitrophenol (PNP), o-nitrophenol (ONP), or p-aminophenol (PAP) as products. Electrochemical detection using stencil-printed carbon electrodes (SPCEs) was found to provide optimal performance on inexpensive and disposable transparency film platforms. Using SPCEs, detection limits for electrochemically active substrates, PNP, ONP, and PAP were determined to be 1.1, 2.8, and 0.5 μM, respectively. A colorimetric paper-based well plate system was developed from a simple cardboard box and smart phone for the detection of PNP and ONP. Colorimetric detection limits were determined to be 81 μM and 119 μM for ONP and PNP respectively. While colorimetric detection methods gave higher detection limits than electrochemical detection, both methods provided similar times to positive bacteria detection. Low concentrations (10

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.

An accurate, rapid, and cost-effective methodology for enzyme inhibitor assays is highly needed for large-scale screening to evaluate the efficacy of drugs at the molecular level. For the first time, we have developed an inkjet printing-based enzyme inhibition assay for the assessment of drug activity using a conventional inkjet printer composed of four cartridges. The methodology is based on the determination of the number of moles of the drug on the printed surface. The number of moles was quantified through the volume of substance ejected onto the printed surface. The volume ejected on the reaction spot was determined from the density of reagent ink solution and its weight loss after printing. A xanthine oxidase (XOD) inhibition assay was executed to quantitatively evaluate antioxidant activities of the drug based on the determination of the number of moles of the drug ejected by inkjet printing. The assay components of xanthine, nitro blue tetrazolium (NBT), superoxide dismutase (SOD)/drug, and XOD were printed systematically on A4 paper. A gradient range of the number of moles of SOD/drug printed on A4 paper could be successfully obtained. Because of the effect of enzyme activity inhibition, incrementally reduced NBT formazan colors appeared on the paper in a number-of-moles-dependent manner. The observed inhibitory mole (IM

This paper reports on the development of a hybrid atmospheric pressure atomic force microscopy/mass spectrometry imaging system utilizing nanothermal analysis probes for thermal desorption surface sampling with subsequent atmospheric pressure chemical ionization and mass analysis. The basic instrumental setup and the general operation of the system were discussed, and optimized performance metrics were presented. The ability to correlate topographic images of a surface with atomic force microscopy and a mass spectral chemical image of the same surface, utilizing the same probe without moving the sample from the system, was demonstrated. Co-registered mass spectral chemical images and atomic force microscopy topographical images were obtained from inked patterns on paper as well as from a living bacterial colony on an agar gel. Spatial resolution of the topography images based on pixel size (0.2 μm × 0.8 μm) was better than the resolution of the mass spectral images (2.5 μm × 2.0 μm), which were limited by current mass spectral data acquisition rate and system detection levels.

A device for measuring human breath ammonia was developed based on a single use, disposable, inkjet printed ammonia sensor fabricated using polyaniline nanoparticles. The device was optimized for sampling ammonia in human breath samples by addressing issues such as variations in breath sample volume, flow rate, sources of oral ammonia, temperature and humidity. The resulting system was capable of measuring ammonia in breath from 40 to 2993 ppbv (r(2 )= 0.99, n = 3) as correlated with photoacoustic laser spectroscopy and correlation in normal human breath samples yielded a slope of 0.93 and a Pearson correlation coefficient of 0.9705 (p0.05, n = 11). Measurement of ammonia in the breath of patients with end-stage kidney disease demonstrated its significant reduction following dialysis, while also correlating well with blood urea nitrogen (BUN) (r = 0.61, p0.01, n = 96). Excellent intraindividual correlations were demonstrated between breath ammonia and BUN (0.86 to 0.96), which demonstrates the possibility of using low cost point of care breath ammonia systems as a noninvasive means of monitoring kidney dysfunction and treatment.

In this work, a radiofrequency identification (RFID) tag with an optical indicator for the measurement of gaseous oxygen is described. It consists of an O2 sensing membrane of PtOEP together with a full electronic system for RFID communication, all printed on a flexible substrate. The membrane is excited by an LED at 385 nm wavelength and the intensity of the luminescence generated is registered by means of a digital color detector. The output data corresponding to the red coordinate of the RGB color space is directly related to the concentration of O2, and it is sent to a microcontroller. The RFID tag is designed and implemented by screen printing on a flexible substrate for the wireless transmission of the measurement to a remote reader. It can operate in both active and passive mode, obtaining the power supply from the electromagnetic waves of the RFID reader or from a small battery, respectively. This system has been fully characterized and calibrated including temperature drifts, showing a high-resolution performance that allows measurement of very low values of oxygen content. Therefore this system is perfectly suitable for its use in modified atmosphere packaging where the oxygen concentration is reduced below 2%. As the reading of the O2 concentration inside the envelope is carried out with an external RFID reader using wireless communication, there is no need for perforations for probes or wires, so the packaging remains completely closed. With the presented device, a limit of detection of 40 ppm and a resolution as low as 0.1 ppm of O2 can be reached with a low power consumption of 3.55 mA.

We demonstrated a minimally destructive two-tier approach for multielement forensic analysis of laser-printed ink. The printed document was first screened using a portable-X-ray fluorescence (XRF) probe. If the results were not conclusive, a laser microprobe was then deployed. The laser probe was based on a two-pulse scheme: the first laser pulse ablated a thin layer of the printed ink; the second laser pulse at 193 nm induced multianalytes in the desorbed ink to fluoresce. We analyzed four brands of black toners. The toners were printed on paper in the form of patches or letters or overprinted on another ink. The XRF probe could sort the four brands if the printed letters were larger than font 20. It could not tell the printing sequence in the case of overprints. The laser probe was more discriminatory; it could sort the toner brands and reveal the overprint sequence regardless of font size while the sampled area was not visibly different from neighboring areas even under the microscope. In terms of general analytical performance, the laser probe featured tens of micrometer lateral resolution and tens to hundreds of nm depth resolution and atto-mole mass detection limits. It could handle samples of arbitrary size and shape and was air compatible, and no sample pretreatment was necessary. It will prove useful whenever high-resolution and high sensitivity 3D elemental mapping is required.

A simple separation and detection system based on an electrochemical potential modulated microchannel (EPMM) device was developed for the first time. The application of alternating current (AC) potential to the microfluidic separation channel walls, which were composed of screen printed carbon electrodes, resulted in the oscillation and fluctuation of analytes and in the formation of a perfect flat flow front. These events resulted in an increase in the effective concentration and in the fine separation of samples. The performance of the EPMM device was examined through the analysis of endocrine disruptors (EDs) and heavy metal ions (HMIs) as model compounds. The analytical parameters that affected the separation and detection of EDs and HMIs were studied in terms of AC amplitude, AC frequency, flow rate, buffer concentration, pH, detection potential, and temperature. The separation efficiency was evaluated through measurements of the theoretical plate number (N), the retention time, and the half-peak width. Linear calibration plots for the detection of EDs and HMIs were obtained between 0.15 and 250.0 nM (detection limit 86.4 ± 2.9 pM) and between 0.01 and 10.0 nM (detection limit 9.5 ± 0.3 pM), respectively. The new device was successfully demonstrated with authentic and real samples.

This study describes a novel method for single-cell analysis and lipid profiling by combining drop-on-demand inkjet cell printing and probe electrospray ionization mass spectrometry (PESI-MS). Through inkjet sampling of a cell suspension, droplets with single cells were generated, precisely dripped onto a tungsten-made electrospray ionization needle, and immediately sprayed under a high-voltage electric field. Lipid fingerprints of single cells were obtained by a mass spectrometry (MS) detector. A homemade magnetic stirring device was applied to the cell suspension reservoir, which controlled the homogeneous distribution of cells in liquid and improved the single-cell-droplet percentage by 43.8%. Eight types of single cells were screened in our platform and further differentiated by principal component analysis based on cellular surface phospholipids. Thus, this study successfully provides a facile method for the direct MS profiling of single-cell lipids by PESI-MS.

Preparation of raw, untreated biological samples remains a major challenge in microfluidics. We present a novel microfluidic device based on the integration of printed circuit boards and an isotachophoresis assay for sample preparation of nucleic acids from biological samples. The device has integrated resistive heaters and temperature sensors as well as a 70 μm × 300 μm × 3.7 cm microfluidic channel connecting two 15 μL reservoirs. We demonstrated this device by extracting pathogenic nucleic acids from 1 μL dispensed volume of whole blood spiked with Plasmodium falciparum. We dispensed whole blood directly onto an on-chip reservoir, and the system's integrated heaters simultaneously lysed and mixed the sample. We used isotachophoresis to extract the nucleic acids into a secondary buffer via isotachophoresis. We analyzed the convective mixing action with micro particle image velocimetry (micro-PIV) and verified the purity and amount of extracted nucleic acids using off-chip quantitative polymerase chain reaction (PCR). We achieved a clinically relevant limit of detection of 500 parasites per microliter. The system has no moving parts, and the process is potentially compatible with a wide range of on-chip hybridization or amplification assays.

In this study, a novel disposable all-solid-state ion-selective electrode using graphene as the ion-to-electron transducer was developed. The graphene film was prepared on screen-printed electrode directly from the graphene oxide dispersion by a one-step electrodeposition technique. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to demonstrate the large double layer capacitance and fast charge transfer of the graphene film modified electrode. On the basis of these excellent properties, an all-solid-state calcium ion-selective electrode as the model was constructed using the calcium ion-selective membrane and graphene film modified electrode. The mechanism about the graphene promoting the ion-to-electron transformation was investigated in detail. The disposable electrode exhibited a Nernstian slope (29.1 mV/decade), low detection limit (10(-5.8) M), and fast response time (less than 10 s). With the high hydrophobic character of graphene materials, no water film was formed between the ion-selective membrane and the underlying graphene layer. Further studies revealed that the developed electrode was insensitive to light, oxygen, and redox species. The use of the disposable electrode for real sample analysis obtained satisfactory results, which made it a promising alternative in routine sensing applications.

We report a new type of microarray, based on glyconanoparticles (GNPs), to study glycan-lectin interactions. GNPs, synthesized by conjugating carbohydrate ligands on silica nanoparticles, were printed on a photoactive surface followed by covalent immobilization by light activation. The GNP microarrays could be probed by lectins labeled with fluorescein as well as fluorescein-doped silica nanoparticles (FSNPs). Results showed that FSNP as the label enhanced the signals for the higher affinity ligands than the lower ones.

A renewable flow cell integrating a microstructured pillar-array filter and a pneumatic microvalve was microfabricated to trap and release beads. A bead-based immunoassay using this device was also developed. This microfabricated device consists of a microfluidic channel connecting to a beads chamber in which the pillar-array filter is built. Underneath the filter, there is a pneumatic microvalve built across the chamber. Such a device can trap and release beads in the chamber by "closing" or "opening" the microvalve. On the basis of the pneumatic microvalve, the device can trap beads in the chamber before performing an assay and release the used beads after the assay. Therefore, this microfabricated device is suitable for "renewable surface analysis". A model analyte, 3,5,6-trichloropyridinol (TCP), was chosen to demonstrate the analytical performance of the device. The entire fluidic assay process, including beads trapping, immuno binding, beads washing, beads releasing, and chemiluminesence signal collection, could be completed in 10 min. The immunoassay of TCP using this microfabricated device showed a linear range of 0.20-70 ng/mL with a limit of detection of 0.080 ng/mL. The device was successfully used to detect TCP spiked in human plasma at the concentration range of 1.0-50 ng/mL, with an analytical recovery of 81-110%. The results demonstrated that this device can provide a rapid, sensitive, reusable, low-cost, and automatic tool for detecting various biomarkers in biological fluids.

Microfluidic cell-based systems have enabled the study of cellular phenomena with improved spatiotemporal control of the microenvironment and at increased throughput. While poly(dimethylsiloxane) (PDMS) has emerged as the most popular material in microfluidics research, it has specific limitations that prevent microfluidic platforms from achieving their full potential. We present here a complete process, ranging from mold design to embossing and bonding, that describes the fabrication of polystyrene (PS) microfluidic devices with similar cost and time expenditures as PDMS-based devices. Emphasis was placed on creating methods that can compete with PDMS fabrication methods in terms of robustness, complexity, and time requirements. To achieve this goal, several improvements were made to remove critical bottlenecks in existing PS embossing methods. First, traditional lithographic techniques were adapted to fabricate bulk epoxy molds capable of resisting high temperatures and pressures. Second, a method was developed to emboss through-holes in a PS layer, enabling creation of large arrays of independent microfluidic systems on a single device without need to manually create access ports. Third, thermal bonding of PS layers was optimized in order to achieve quality bonding over large arrays of microsystems. The choice of materials and methods was validated for biological function in two different cell-based applications to demonstrate the versatility of our streamlined fabrication process.

We report on the use of photonic crystal surfaces as a high-sensitivity platform for detection of a panel of cancer biomarkers in a protein microarray format. The photonic crystal surface is designed to provide an optical resonance at the excitation wavelength of cyanine-5 (Cy5), thus providing an increase in fluorescent intensity for Cy5-labeled analytes measured with a confocal microarray scanner, compared to a glass surface. The sandwich enzyme-linked immunosorbent assay (ELISA) is undertaken on a microarray platform to undertake a simultaneous, multiplex analysis of 24 antigens on a single chip. Our results show that the resonant excitation effect increases the signal-to-noise ratio by 3.8- to 6.6-fold, resulting in a decrease in detection limits of 5–90%, with the exact enhancement dependent upon the antibody-antigen interaction. Dose-response characterization of the photonic crystal antibody microarrays shows the capability to detect common cancer biomarkers in the < 2 pg/ml concentration range within a mixed sample.

This paper describes the first approach at combining paper microfluidics with electrochemiluminescent (ECL) detection. Inkjet printing is used to produce paper microfluidic substrates which are combined with screen-printed electrodes (SPEs) to create simple, cheap, disposable sensors which can be read without a traditional photodetector. The sensing mechanism is based on the orange luminescence due to the ECL reaction of tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3)(2+)) with certain analytes. Using a conventional photodetector, 2-(dibutylamino)ethanol (DBAE) and nicotinamide adenine dinucleotide (NADH) could be detected to levels of 0.9 μM and 72 μM, respectively. Significantly, a mobile camera phone can also be used to detect the luminescence from the sensors. By analyzing the red pixel intensity in digital images of the ECL emission, a calibration curve was constructed demonstrating that DBAE could be detected to levels of 250 μM using the phone.

This Technical Note demonstrates a simple method based on flexographic printing of polystyrene to form liquid guiding boundaries and layers on paper substrates. The method allows formation of hydrophobic barrier structures that partially or completely penetrate through the substrate. This unique property enables one to form very thin fluidic channels on paper, leading to reduced sample volumes required in point-of-care diagnostic devices. The described method is compatible with roll-to-roll flexography units found in many printing houses, making it an ideal method for large-scale production of paper-based fluidic structures.

We report a technique that utilizes surface plasmon resonance dispersion as a mechanism to provide multicolor contrast for imaging thin molecular films. Illumination of gold surfaces with p-polarized white light in the Kretschmann configuration produces distinct reflected colors due to excitation of surface plasmons and the resulting absorption of specific wavelengths from the source light. In addition, these colors transform in response to the formation of thin molecular films. This process represents a simple detection method for distinguishing between films of varying thickness in sensor applications. As an example, we interrogated a protein microarray formed by a commercial drop-on-demand chemical ink jet printer. Submonolayer films of a test protein (bovine serum albumin) were readily detected by this method. Analysis of the dispersion relations and absorbance sensitivities illustrate the performance and characteristics of this system. Higher detection sensitivity was achieved at angles where red wavelengths coupled to surface plasmons. However, improved contrast and spatial resolution occurred when the angle of incidence was such that shorter wavelengths coupled to the surface plasmons. Simplified optics combined with the robust microarray printing platform are used to demonstrate the applicability of this technique as a rapid and versatile, high-throughput tool for label-free detection of adsorbed films and macromolecules.

We report on a quartz pin that can be interfaced easily to existing pin printers. The new pin surface can be reversibly derivatized using silanization chemistry, allowing one to reliably print a wide variety of liquid solutions. Feature sizes as small as 9 microm can be produced with the new pin, allowing one to readily create microarrays with a feature density approaching 10(6) spots/cm2.

A paper test card has been engineered to perform an iodometric titration, an application that requires storage and mixing on demand of several mutually incompatible reagents. The titration is activated when a user applies a test solution to the test card: the dried reagents are reconstituted and combined through a surface-tension-enabled mixing (STEM) mechanism. The device quantifies 0.8-15 ppm of iodine atoms from iodate in aqueous solutions. This is useful, for example, to quantify iodine levels in fortified salt. A blinded internal laboratory validation established the accuracy as 1.4 ppm I and the precision as 0.9 ppm I when the test card was read by newly trained users. Using computer software to process images, the accuracy and precision both improved to 0.9 ppm I. The paper card can also detect substandard β lactam antibiotics using an iodometric back-titration. When used to quantify amoxicillin, good distinction is achieved between solutions that differ by 0.15 mg/mL over a working range of 0-0.9 mg/mL. The test card was designed to meet the World Health Organization ASSURED criteria for use in low resource settings, where laboratory-based analytical procedures are often not available.

Digital microfluidics is a fluid manipulation technique in which discrete droplets are actuated on patterned arrays of electrodes. Although there is great enthusiasm for the application of this technique to chemical and biological assays, development has been hindered by the requirement of clean room fabrication facilities. Here, we present a new fabrication scheme, relying on microcontact printing (microCP), an inexpensive technique that does not require clean room facilities. In microCP, an elastomeric poly(dimethylsiloxane) stamp is used to deposit patterns of self-assembled monolayers onto a substrate. We report three different microCP-based fabrication techniques: (1) selective etching of gold-on-glass substrates; (2) direct printing of a suspension of palladium colloids; and (3) indirect trapping of gold colloids from suspension. In method 1, etched gold electrodes are used for droplet actuation; in methods 2 and 3, colloid patterns are used to seed electroless deposition of copper. We demonstrate, for the first time, that digital microfluidic devices can be formed by microCP and are capable of the full range of digital microfluidics operations: dispensing, merging, motion, and splitting. Devices formed by the most robust of the new techniques were comparable in performance to devices formed by conventional methods, at a fraction of the fabrication time. These new techniques for digital microfluidics device fabrication have the potential to facilitate expansion of this technology to any research group, even those without access to conventional microfabrication tools and facilities.

We report the fabrication of three-dimensional living-cell microarrays via pin-printing of soft sol-gel-derived silica materials containing bacterial cells. Bacterial cells entrapped in the silica-glycerol microarray spots can express reporter genes and produce strong fluorescence signals. The signals responded to the presence and concentration of inducers or repressors as expected, indicating that the entrapped cells remained metabolically active. Microscopic imaging of individual microarray spots at different culture times suggests that the entrapped cells can grow and divide, phenomena further confirmed by experiments in bulk sol-gel materials that demonstrated the increases of entrapped cell density and fluorescence during incubation in culture media. The cell microarrays can also be printed into 96-well glass bottom microtiter plates in a multiplexed manner, and the fluorescence signals generated were able to quantitatively and selectively respond to the concentration of inducers, thus demonstrating the potential for multitarget biosensing and high-throughput/high-content cell-based screening. The signal levels of bacterial cells in silica were significantly higher than those in alginate arrays, presumably due to viability of the entrapped cells in silica sol-gels. Microarray stability assays proved that the entrapped cells retained their physiological activity after storage for four weeks. Given that a large number of fluorescent and luminescent protein-based cell assays have been developed, the reporter gene living-cell microarrays demonstrated in this paper are expected to be applicable to a wide variety of research areas ranging from bioanalysis and chemical biology to drug discovery and probing of cell-material interactions.

This paper describes a colorimetric sensor array for the discrimination of volatile amines. Analyte discrimination is achieved by combining two functional elements: (1) a "chemical class-selective" single chromogenic sensing dye with selectivity for amines in general, encapsulated into (2) polymer nanoparticles with different polarities. The resulting array has the ability to distinguish one closely related amine from another, relying on a polarity-based approach. In order to achieve reproducible, cost efficient, and flexible sensor array fabrication with the potential for mass production, inkjet-printing technology combined with standard copy paper as a sensor substrate is applied. Printing of 6 types of inks, which are prepared by mixing two dye encapsulating nanoparticles of different polarity in different mixture ratios, results in a colorimetric sensor array with a polarity gradient. Seven primary amines with increasing alkyl chain lengths have been selected to demonstrate the performance of the sensor array. The RGB color differences (ΔR, ΔG, ΔB) of the sensor array spots before and after gas exposure were analyzed by principal component analysis (PCA) and agglomerative hierarchical clustering (AHC) analysis. Under the selected measurement conditions, results of PCA and AHC analysis indicated high discrimination ability with high reproducibility of the sensor array down to amine concentrations of 50 ppm. The discrimination ability was maintained at relative humidities between 10% and 80%. Furthermore, the sensor array showed no significant response to common volatile organic compounds, confirming the high selectivity toward amines. This is, to the best of our knowledge, the first report of a colorimetric sensor array with selectivity for a specific chemical class of analytes and the ability to discriminate compounds of the same class, which is obtained by simply mixing two types of single dye-encapsulating polymer nanoparticles.

A new type of paper based bioassay for the colorimetric detection of phenolic compounds including phenol, bisphenol A, catechol and cresols is reported. The sensor is based on a layer-by-layer (LbL) assembly approach formed by alternatively depositing layers of chitosan and alginate polyelectrolytes onto filter paper and physically entrapping the tyrosinase enzyme in between these layers. The sensor response is quantified as a color change resulting from the specific binding of the enzymatically generated quinone to the multilayers of immobilized chitosan on the paper. The color change can be quantified with the naked eye but a digitalized picture can also be used to provide more sensitive comparison to a calibrated color scheme. The sensor was optimized with respect to the number of layers, pH, enzyme, chitosan and alginate amounts. The colorimetric response was concentration dependent, with a detection limit of 0.86 (±0.1) μg/L for each of the phenolic compounds tested. The response time required for the sensor to reach steady-state color varied between 6 and 17 min depending on the phenolic substrate. The sensor showed excellent storage stability at room temperature for several months (92% residual activity after 260 days storage) and demonstrated good functionality in real environmental samples. A procedure to mass-produce the bioactive sensors by inkjet printing the LbL layers of polyelectrolyte and enzyme on paper is demonstrated.

A new device to perform spectroelectrochemical measurements in the UV/visible spectral region using screen-printed electrodes has been developed. Neurotransmitter dopamine has been selected as a proof of concept of the capabilities of the new device. The results obtained have allowed us both to study the oxidation mechanism of dopamine and to carry out the spectroelectrochemical detection of this neurotransmitter. Differences in dopamine oxidation mechanism have been observed depending on the initial concentration. Thus, dopamine concentrations lower than 10(-3) M led to a higher generation of dopaminochrome and its derivatives with a band centered at 305 nm, which was the best wavelength to determine dopamine spectrophotometrically at these concentrations. However, if dopamine concentration is higher than 10(-3) M, dopaminoquinone is stable enough to use its maximum of absorbance, 395 nm, to detect this neurotransmitter. Dopamine concentration can also be calculated from the electrochemical data in spectroelectrochemistry, the results being comparable to that obtained from spectroscopic data. Comparison between spectrophotometric and electrochemical determinations demonstrates that the two methods measure this analyte indistinctively, proving that spectroelectrochemistry represents an autovalidated technique. Partial least-squares regression has also been used, obtaining good results in the full dopamine concentration range. Finally, as spectroelectrochemistry is an intrinsically trilinear technique, PARAFAC has been used to study the effect of probable interfering species.

Wafer scale (cm(2)) arrays and networks of nanochannels were created in polydimethylsiloxane (PDMS) from a surface pattern of electrodeposited gold nanowires in a master-replica process and characterized with scanning electron microscopy (SEM), atomic force microscopy (AFM), and fluorescence imaging measurements. Patterns of gold nanowires with cross-sectional dimensions as small as 50 nm in height and 100 nm in width were prepared on silica substrates using the process of lithographically patterned nanowire electrodeposition (LPNE). These nanowire patterns were then employed as masters for the fabrication of inverse replica nanochannels in a special formulation of PDMS. SEM and AFM measurements verified a linear correlation between the widths and heights of the nanowires and nanochannels over a range of 50 to 500 nm. The PDMS replica was then oxygen plasma-bonded to a glass substrate in order to create a linear array of nanofluidic channels (up to 1 mm in length) filled with solutions of either fluorescent dye or 20 nm diameter fluorescent polymer nanoparticles. Nanochannel continuity and a 99% fill success rate was determined from the fluorescence imaging measurements, and the electrophoretic injection of both dye and nanoparticles in the nanochannel arrays was also demonstrated. Employing a double LPNE fabrication method, this master-replica process was also used to create a large two-dimensional network of crossed nanofluidic channels.

A simple approach to the mass production of nanoporous gold electrode arrays on cellulose membranes for electrochemical sensing of oxygen using ionic liquid (IL) electrolytes was established. The approach, combining the inkjet printing of gold nanoparticle (GNP) patterns with the self-catalytic growth of these patterns into conducting layers, can fabricate hundreds of self-designed gold arrays on cellulose membranes within several hours using an inexpensive inkjet printer. The resulting paper-based gold electrode arrays (PGEAs) had several unique properties as thin-film sensor platforms, including good conductivity, excellent flexibility, high integration, and low cost. The porous nature of PGEAs also allowed the addition of electrolytes from the back cellulose membrane side and controllably produced large three-phase electrolyte/electrode/gas interfaces at the front electrode side. A novel paper-based solid-state electrochemical oxygen (O(2)) sensor was therefore developed using an IL electrolyte, 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF(6)). The sensor looked like a piece of paper but possessed high sensitivity for O(2) in a linear range from 0.054 to 0.177 v/v %, along with a low detection limit of 0.0075% and a short response time of less than 10 s, foreseeing its promising applications in developing cost-effective and environment-friendly paper-based electrochemical gas sensors.

Mucins are the principal components of mucus, and mucin glycosylation has important roles in defense, microbial adhesion, immunomodulation, inflammation, and cancer. Mucin expression and glycosylation are dynamic, responding to changes in local environment and disease. Potentially hundreds of heterogeneous glycans can substitute one mucin molecule, and it is difficult to identify biologically accessible glyco-epitopes. Thirty-seven mucins, from the reproductive and gastrointestinal (GI) tracts of six species (bovine, ovine, equine, porcine, chicken, and deer) and from two human-derived cell lines, were purified. Following optimization of mucin printing and construction of a novel mucin microarray, the glycoprofiles of the whole mucins on the microarray were compared using a panel of lectins and one antibody. Accessible glyco-motifs of GI mucins varied according to species and localization of mucin origin, with terminal fucose, the sialyl T-antigen, and N-linked oligosaccharides identified as potentially important. The occurrence of T- and sialyl T-antigen varied in bovine and ovine reproductive tract mucins, and terminal N-acetylgalactosamine (GalNAc) and sulfated carbohydrates were detected. This study introduces natural mucin microarrays as an effective tool for profiling mucin glyco-epitopes and highlights their potential for discovery of biologically important motifs in bacterial-host interactions and fertility.

International audience; Molecularly imprinted polymer (MIP) droplets were printed using a pipet or a nano fountain pen on surface-enhanced Raman scattering (SERS)-active surfaces, to directly monitor the uptake and release of a template molecule, the beta-blocking drug propranolol, by SERS. The monitored SERS bands can be related to the template, allowing for its detection but also identification in the MIP. This is an advantage if the technique is to be used during the development phase of MIPs as microstructures, but equally for the readout of MIP-based biochips.

Careful matrix deposition on tissue samples for matrix-assisted laser desorption/ionization (MALDI) is critical for producing reproducible analyte ion signals. Traditional methods for matrix deposition are often considered an art rather than a science, with significant sample-to-sample variability. Here we report an automated method for matrix deposition, employing a desktop inkjet printer ($200) with 5760 x 1440 dpi resolution and a six-channel piezoelectric head that delivers 3 pL/drop. The inkjet printer tray, designed to hold CDs and DVDs, was modified to hold microscope slides. Empty ink cartridges were filled with MALDI matrix solutions, including DHB in methanol/water (70:30) at concentrations up to 40 mg/mL. Various samples (including rat brain tissue sections and standards of small drug molecules) were prepared using three deposition methods (electrospray, airbrush, inkjet). A linear ion trap equipped with an intermediate-pressure MALDI source was used for analyses. Optical microscopic examination showed that matrix crystals were formed evenly across the sample. There was minimal background signal after storing the matrix in the cartridges over a 6-month period. Overall, the mass spectral images gathered from inkjet-printed tissue specimens were of better quality and more reproducible than from specimens prepared by the electrospray and airbrush methods.