Microarray-based kinase inhibition assay by gold nanoparticle probes

We report on the development of a new class of kinase microarray for the detection of kinase inhibition based on marking peptide phosphorylation/biotinylation events by attachment of gold nanoparticles followed by silver deposition for signal enhancement. The alpha-catalytic subunit of cyclic adenosine 5'-monophosphate-dependent protein kinase (PKA), and its well-known substrate, kemptide, were used for the purpose of monitoring phosphorylation and inhibition. As expected, highly selective inhibition of PKA is demonstrated with the four inhibitors: H89, HA1077, mallotoxin, and KN62. Furthermore, an inhibition assay demonstrates the ability to detect kinase inhibition as well as derive IC50 (half-maximal inhibitory concentration) plots.     

Label-free fluorescent detection of protein kinase activity based on the aggregation behavior of unmodified quantum dots

Herein, we present a novel label-free fluorescent assay for monitoring the activity and inhibition of protein kinases based on the aggregation behavior of unmodified CdTe quantum dots (QDs). In this assay, cationic substrate peptides induce the selective aggregation of unmodified QDs with anionic surface charge, whereas phosphorylated peptides do not. Phosphorylation by kinase alters the net charge of peptides and subsequently inhibits the aggregation of unmodified QDs, causing an enhanced fluorescence with a 45 nm blue-shift in emission and a yellow-to-green emission color change. Hence the fluorescence response allows this QD-based method to easily probe kinase activity by a spectrometer or even by the naked eye. The feasibility of the method has been demonstrated by sensitive measurement of the activity of cAMP-dependent protein kinase (PKA) with a low detection limit (0.47 mU μL(-1)). On the basis of the fluorescence response of QDs on the concentration of PKA inhibitor H-89, the IC(50) value, the half maximal inhibitory concentration, was estimated, which was in agreement with the literature value. Moreover, the system can be applicable to detect the Forskolin/3-isobutyl-1-methylxantine (IBMX)-stimulated activation of PKA in cell lysate. Unlike the existing QD-based enzyme activity assays in which the modification process of QDs is essential, this method relies on unmodified QDs without the requirement of peptide labeling and QDs' modification, presenting a promising candidate for cost-effective kinase activity and inhibitor screening assays.     

Aptameric peptide for one-step detection of protein kinase

Protein kinases are significant regulators in the cell signal pathway, and it is difficult to achieve quick kinase detection because traditional kinase assays normally rely on a time-consuming kinase phosphorylation process. Herein, we present a novel one-step strategy to detect protein kinase by using a kinase-specific aptameric peptide-functionalized quartz crystal microbalance (QCM) electrode, in which the detection can be finished in less than 10 min. A peptide kinase inhibitor (IP(20)) was used as the aptameric peptide because of its selective and strong interaction with the target protein kinase (cyclic adenosine monophosphate-dependent protein kinase A, PKA), high stability, and ease of inexpensive synthesis, presenting a new direct recognition element for kinase. The aptameric peptide was immobilized on the Au-coated quartz electrode through dual-thiol anchoring and the binding of His-tagged peptide with a nitrilotriacetic acid/Ni(II) complex, fabricating a highly specific and stable detection platform. The interaction of aptameric peptide with kinase was monitored with the QCM in real time, and the concentration of protein kinase was sensitively measured by the frequency response of the QCM with the low detection limit for PKA at 0.061 mU μL(-1) and a linear range from 0.64 to 22.33 mU μL(-1). This method is rapid and reagentless and does not require a phosphorylation process. The versatility of our aptameric peptide-based strategy has also been demonstrated by the application in kinase assay using electrochemical impedance spectroscopy. Moreover, this method was successfully applied to detect the forskolin/3-isobutyl-1-methylxanthine-stimulated activation of PKA in cell lysate.     

Microarray-based detection of protein binding and functionality by gold nanoparticle probes

We report a microarray format for the detection of proteins and protein functionality (kinase activity) based on marking either specific antibody-protein binding or peptide phosphorylation events by attachment of gold nanoparticles followed by silver deposition for signal enhancement. The attachment of the gold nanoparticles is achieved by standard avidin-biotin chemistry. The detection principle is resonance light scattering. Highly selective recognition of standard proteins (proteins A and G) down to 1 pg/mL for proteins in solution and 10 fg for proteins on the microarray spots is demonstrated. Enzyme activity of the kinase (PKA) is detected with high specificity down to a limit of 1 fg for an established peptide substrate (kemptide) on the microarray spots. Kinase inhibition by the inhibitor (H89) is shown, demonstrating the potential for high-throughput screening for inhibitors.     

Highly sensitive electrogenerated chemiluminescence biosensor in profiling protein kinase activity and inhibition using gold nanoparticle as signal transduction probes

A novel electrogenerated chemiluminescence (ECL) biosensor using gold nanoparticles as signal transduction probes was described for the detection of kinase activity. The gold nanoparticles were specifically conjugated to the thiophosphate group after the phosphorylation process in the presence of adenosine 59-[c-thio] triphosphate (ATP-s) cosubstrate. Due to its good conductivity, large surface area, and excellent electroactivity to luminol oxidization, the gold nanoparticles extremely amplified the ECL signal of luminol, offering a highly sensitive ECL biosensor for kinase activity detection. Protein kinase A (PKA), an important enzyme in regulation of glycogen, sugar, and lipid metabolism in the human body, was used as a model to confirm the proof-of-concept strategy. The as-proposed biosensor presented high sensitivity, low detection limit of 0.07 U mL(-1), wide linear range (from 0.07 to 32 U mL(-1)), and excellent stability. Moreover, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent ECL signal, the half-maximal inhibition value IC(50) of ellagic acid, a PKA inhibitor, was estimated, which was in agreement with those characterized with the conventional kinase assay. While nearly no ECL signal change can be observed in the presence of Tyrphostin AG1478, a tyrosine kinase inhibitor, but not PKA inhibitor, shows its excellent performance in kinase inhibitor screening. The simple and sensitive biosensor is promising in developing a high-through assay of in vitro kinase activity and inhibitor screening for clinic diagnostic and drug development.     

Use of capillary electrophoresis and endogenous fluorescent substrate to monitor intracellular activation of protein kinase A

Here we demonstrate for the first time the use of an endogenous multiphosphorylatable substrate for monitoring the intracellular activation of kinase with capillary electrophoresis. First, we devised a novel PCR-based strategy for controlled generation of short multirepeat DNA sequences and applied this method to generate a green fluorescence protein (GFP)-tagged protein substrate containing eight phosphorylation sites for protein kinase A (PKA). The protein substrate was transiently expressed in C2C12 rat myoblast cells, and intracellular PKA was then activated by adding [8]-bromo-cyclic AMP to the cell culture medium. Phosphorylated product and nonphosphorylated substrate present in the crude cell extract were separated by capillary zone electrophoresis and detected with laser-induced fluorescence of the GFP tag. The identities of two electrophoretic peaks were confirmed by both phosphorylation of the substrate and dephosphorylation of the product in vitro. The proposed method was applied to monitoring the activation of PKA in single myoblast cells. It advantageously allowed us to avoid microinjection of the substrate, the procedure that is both hard to perform and excessively invasive when applied to small mammalian cells.     

Improved sensitivity for phosphopeptide mapping using capillary column HPLC and microionspray mass spectrometry: comparative phosphorylation site mapping from gel-derived proteins

Reversible protein phosphorylation regulates many cellular processes. Understanding how phosphorylation controls a given pathway usually involves specific knowledge of which amino acid residues are phosphorylated on a given protein. This is often a nontrivial task. In addition to the difficulties involved in purifying sufficient amounts of any given protein, most phosphoproteins contain multiple, substoichiometric sites of phosphorylation. In this paper, we describe substantial improvements made to our previously reported multidimensional electrospray MS-based phosphopeptide mapping technique that have resulted in a 20-fold increase in sensitivity for the overall process. Chief among these improvements are the incorporation of capillary chromatography and a microionspray source for the mass spectrometer into the first dimension of the analysis. In the first dimension of the process, phosphopeptides present in the proteolytic digest of a protein are selectively detected and collected into fractions during on-line LC/ESMS, which monitors for phosphopeptide specific marker ions. The phosphopeptide containing fractions are then analyzed in the second dimension by either MALDI-PSD or nano-ES with precursor ion scanning. The relative merits and limitations of these two techniques for phosphopeptide detection are demonstrated. The enhancement in sensitivity of the method under the new experimental conditions makes it suitable for phosphorylation mapping (from selective detection through sequencing) on gel-separated phosphoproteins where the level of phosphorylation at any given site is <200 fmol. Furthermore, this method detects serine, threonine, and tyrosine phosphorylation equally well. We have successfully employed this new configuration to map 11 in vivo sites of phosphorylation on the Saccharomyces cerevisiae protein kinase YAK1. YAK1 peptides containing all five YAK1 PKA consensus sites are phosphorylated, suggesting that YAK1 is an in vivo substrate for PKA. In addition, four peptides containing cdk sites and the autophosphorylation site at Tyr530 were found to be phosphorylated. Because the first dimension of this method generates a phosphorylation profile that can be used for a semiquantitative evaluation of site specific phosphoxylation, we evaluated its ability to detect site-specific changes in the phosphorylation profile of a protein in response to altered cellular conditions. This comparative phosphopeptide mapping strategy allowed us to detect a change in phosphorylation stoichiometry on the motor protein myosin-V in response to treatment with either mitotic or interphase Xenopus egg extracts and to identify the single functionally significant phosphorylation site that regulates myosin-V cargo binding.     

Label-free electrical sensing of small-molecule inhibition on tyrosine phosphorylation

Protein tyrosine kinases (PTKs) play a central role in human carcinogenesis and have emerged as the promising new targets. Small-molecule inhibitors of PTKs have shown impressive anticancer effects and are rapidly entering the clinic. PTK assays allow for high-throughput identification of small-molecule inhibitors. However, current methods of detecting kinase activity require the use of radioisotopes or expensive reagents; such as fluorescently labeled antibodies. We have developed a novel label-free approach for the quantitative detection of peptide tyrosine (Tyr) phosphorylation using the electrochemical oxidation current signal of Tyr. When the phosphorylation is achieved, the phosphorylated Tyr (Tyr-P) cannot be oxidized at approximately 0.65 V. However, when the phosphorylation is successfully inhibited using a small molecule, Tyr can be oxidized and result in a high current response on a multiwalled carbon nanotube-modified screen-printed carbon electrode. We determined the activity of cellular-sarcoma (c-Src) nonreceptor PTK, p60(c-Src), in combination with its highly specific substrate peptide, Raytide. Tyr kinase reactions were also performed in the presence of a well-defined small-molecule inhibitor, 4-amino-5-(4-chlorophenyl)-7- (tert-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Based on the dependency of Tyr oxidation signal on inhibitor concentration, IC50 value, half-maximal inhibition of the inhibitor, was estimated as 5 nM for PP2. Our label-free electrochemical method is a promising candidate for pharmaceutical research and development in screening small-molecule inhibitors of PTKs.     

Electrical Readout of Protein Microarrays on Regular Glass Slides

A new approach for the electrical readout of microarrays prepared on regular glass slides, using an array of impedimetric transducers (interdigitated electrodes, IDEs) is presented in this work. Impedance detection relies on the use of a urease-labeled immunoassay scheme. Urease is able to produce an increase in conductivity by hydrolysis of the urea substrate, which is measured with the IDEs and directly related to the amount of target analyte. Unlike previous electrical microarrays, the assay does not take place on top of the transducers but on a regular glass slide, which may enable the development of compact multiplexed analytical systems with lower cost per assay. A droplet of solution with the enzymatic substrate is deposited on each transducer of the array, and the microarray is positioned at a short distance (300 μm) so that each droplet wets one transducer and one spot of the microarray. This procedure allows reusing the transducer array for readout of a virtually unlimited number of microarrays. A microarray based on an immunoassay for the detection of a mouse generic protein in a concentration range from 0.03 to 30 μg mL(-1) was carried out to assess the performance of the electrical readout approach. A sigmoid response with a limit of detection of 0.1 μg mL(-1) and a dynamic range of 1 order of magnitude was obtained. A comparative study was also carried out with two well established analytical procedures. First, the urease-based immunoassay was tested in a 96 well microtiter plate using phenol red pH indicator and absorbance detection. Second, the microarray was carried out using the same target protein concentration range but applying a Cy3 label and fluorescence detection. Both assays allowed for the validation of the performance of the presented electrical readout system.     

Enzyme colorimetric assay using unmodified silver nanoparticles

Colorimetric assay based on the unique surface plasmon resonance properties of metallic nanoparticles has received considerable attention in bioassay due to its simplicity, high sensitivity, and low cost. Most of colorimetric methods previously reported employed gold nanoparticles (GNPs) as sensing elements. In this work, we develop a sensitive, selective, simple, and label-free colorimetric assay using unmodified silver nanoparticle (AgNP) probes to detect enzymatic reactions. Enzymatic reactions concerning adenosine triphosphate (ATP) dephosphorylation by calf intestine alkaline phosphatase (CIAP) and peptide phosphorylation by protein kinase A (PKA) were studied. In the absence of the enzymes, unreacted ATP could protect AgNPs from salt-induced aggregation, whereas in the presence of the enzymes, the reaction product of ATP (i.e., adenosine for CIAP and ADP for PKA) could not. Via our method, dephosphorylation and phosphorylation could be readily detected by the color change of AgNPs, with a detection limit of 1 unit/mL for CIAP and a detection limit of 0.022 unit/mL for PKA. More importantly, the enzymatic inhibition by inhibitors and enzymatic activity in complex biological fluids could also be realized. This work is an important step toward a colorimetric assay using AgNPs and might provide a promise for enzyme assay in realistically complex systems and for screening of different enzyme inhibitors in future.     

Real-time protein kinase assay

We report a novel, real-time fluorogenic kinase assay. The peptide substrates are synthesized with a fluorescent dye and a hydrocarbon tail. The substrate self-assembles into micelles, increasing the local concentration of the dye and quenching its fluorescence. Upon phosphorylation, the fluorescence intensity increases 4-6-fold due to micelle reorganization. Both dynamic light scattering data and cryoelectron microscope images show that the size and the shape of the phosphopeptide micelles are significantly different from micelles of substrate peptide. The system provides a robust fluorescence increase in a real-time protein kinase assay. Unlike other fluorogenic systems, the fluorophore may be distant from the serine, threonine, or tyrosine that is phosphorylated. Assays for several kinases, including PKA, PKC, p38, MAPKAP K2, akt, Erk1, and src-family kinases, have been developed. IC(50) values of inhibitors for PKC betaII determined with this technology are consistent with published values. The utility of this assay to high-throughput screening was demonstrated with Sigma's LOPAC library, a collection of 640 compounds with known biological activities, and satisfactory results were obtained.     

A sol-gel-derived acetylcholinesterase microarray for nanovolume small-molecule screening

A fluorimetric acetylcholinesterase (AChE) assay was developed and characterized both in solution and with the enzyme entrapped in sol-gel-derived silica. The assay is based on a disulfide-thiol interchange reaction between the intramolecularly quenched dimeric dye BODIPY FL l-cystine and thiocholine generated by the AChE-catalyzed hydrolysis of acetylthiocholine (ATCh), which results in a brightly fluorescent monomeric product owing to the cleavage of the disulfide-coupled form of the dye. The new assay was validated by comparison with the Ellman assay performed under parallel conditions and was used in both kinetic and end point assays. The assay was extended to the fabrication of functional AChE microarrays using contact pin-printing of sol-gel-derived silica. A total of 392 sol-gel formulations were screened for gelation times and 192 of these were further evaluated for array fabrication on four different surfaces using a factor analysis approach. Of these, 66 sol-gel/surface combinations produced robust microarrays, while 26 sol-gel/surface combinations were identified that could produce highly active AChE microarrays. The Z' factor for the on-array assay using an optimal sol-gel/surface combination, which considers both signal variability and difference in signals between positive and negative controls, was determined to be 0.60, which is above the minimum level required for applicability to screening. By overprinting nanoliter volumes of solutions containing the dye, ATCh, and potential inhibitors, these microarrays could be used to screen two libraries of small molecules, one composed of newly synthesized alkaloids and another consisting of ～1000 known bioactive compounds, both as discrete compounds and mixtures thereof, for activity against AChE. IC(50) values were obtained on microarrays for compounds showing significant inhibitory activity, demonstrating the utility of arrays for quantitative inhibition assays.     

A flow injection kinase assay system based on time-resolved fluorescence resonance energy-transfer detection in the millisecond range

A flow injection analysis (FIA) system for biochemical assays using time-resolved fluorescence resonance energy transfer (TR-FRET) in the millisecond time scale was developed. As a model system, we studied a kinase assay, measuring the phosphorylation of poly(GT)-biotin (substrate) by a receptor tyrosine kinase (epidermal growth factor receptor). A streptavidin labeled with XL665 (SA-XL665)-the acceptor-was coupled to the biotin moiety, and an antiphosphotyrosine antibody labeled with europium cryptate (Ab-EuK)-the donor-was coupled to the phosphorylated tyrosine group(s). Long-lived FRET can only occur if the substrate is successfully phosphorylated. For the time-resolved detection of such long-lived luminescence phenomena in a flow system, the repetition rate of the excitation source plays a crucial role. Good results were obtained for a small-sized commercially available quadrupled Nd:YAG laser emitting at 266 nm with a repetition rate of 7.8 kHz and a pulse width of 0.3 ns. The long-lived emissions of the donor at 625 nm and that of the acceptor at 665 nm were monitored simultaneously with two photomultipliers, using a delay time of 50 micros and a gate time of 75 micros to exclude background fluorescence interferences. In the FIA experiments, the Ab-EuK concentration was 6 nM and the substrate concentration and SA-XL665 concentrations were 7 nM. By monitoring the intensity changes at 625 and 665 nm, the inhibition of tyrosine kinase by tyrphostin AG1478 was studied and an IC(50) value of 5.1 +/- 0.4 nM obtained.     

Analysis of CheA histidine phosphorylation and its influence on protein stability by high-resolution element and electrospray mass spectrometry

A combination of electrospray mass spectrometry (ESI-MS) and element mass spectrometry (ICPMS) with phosphorus detection was used to characterize histidine phosphorylation (His-48) of the chemotaxis protein CheA quantitatively. The phosphorylation at His-48 was found to be responsible for a stabilization of the protein. For this investigation, the acceptor domain and the kinase domain of the bacterial chemotaxis protein CheA were recombinantly expressed as single proteins. Using in vitro kinase assay conditions, the acceptor domain CheA-H was phosphorylated by the kinase domain CheA-C. The degree of histidine phosphorylation was determined by nanoelectrospray mass spectrometry of intact CheA-H, and was found to be limited to a maximum value of approximately 50%. The site specificity of CheA-H phosphorylation was controlled by nanoESI-MS/MS of the [M + 16H](16+) ion of intact (pHis)-CheA-H and allowed localization of the pHis residue to the region between residues 32 and 86, containing candidates His-48 and His-67, for which His-48 phosphorylation has been described. Analysis of the tryptic digest of in vitro histidine-phosphorylated CheA-H by capillary chromatography coupled to ESI-MS and to ICPMS with phosphorus detection revealed a truncated (pHis)-CheA-H protein as the only phosphorus-containing analyte. Since the truncated (pHis)-CheA-H in the digest was found to exhibit a higher degree of phosphorylation than could be generated by in vitro phosphorylation without trypsin treatment, it is concluded that histidine phosphorylation at His-48 strongly interferes with structural properties of the CheA-H domain in particular with respect to proteolytic degradation by trypsin.     

Air-stable G protein-coupled receptor microarrays and ligand binding characteristics

This paper described novel strategies to achieve air-stable G protein-coupled receptor (GPCR) microarrays and the uses of the microarrays for ligand profiling. Specifically, GPCR cell membrane fragments were suspended in a buffered solution containing bovine serum albumin (BSA) and disaccharide sucrose or trehalose and used for fabricating GPCR microarrays. During the array fabrication and postfabrication processes, BSA molecules were found to effectively form packed layer(s) surrounding the GPCR membranes immobilized onto the predetermined printing area, thereby stabilizing the membrane microspots. The use of disaccharides was shown to protect the integrity and functionality of GPCR microarrays from the typical deterioration of the membranes when fabricated and stored under dry conditions. To utilize the ability of fluorescence technology for multichannel detection as well as to maximize the capability of GPCR microarrays for multiplexed binding assays, several fluorescently labeled ligands were synthesized and optimized for multiplexing binding assays. A schematic microarray of five GPCRs had been used as a model for characterizing the association and dissociation rate constants of labeled ligands binding to their respective receptors in the microarrays. Interestingly, distinct receptor-ligand interactions exhibited different dependence on the type of pH reagent as well as the species and concentration of cations used in a binding assay buffered solution. The potential mechanisms and implications for the uses of air-stable GPCR microarrays were discussed.     

Development of a peptidase-resistant substrate for single-cell measurement of protein kinase B activation

An iterative design strategy using three criteria was utilized to develop a peptidase-resistant substrate peptide for protein kinase B. Libraries of peptides possessing non-native amino acids were screened for time to 50% phosphorylation, degradation half-life within a lysate, and appearance of a dominant fragment. The lead peptide possessed a half-life of 92 ± 7 and 16 ± 2 min in HeLa and LNCaP cytosolic lysates, respectively, representing a 4.6- and 2.7-fold lifetime improvement over that of the starting peptide. The redesigned peptide possessed a 4.5-fold improvement in phosphorylation efficiency compared to the starting peptide. The same peptide fragments were formed when the lead peptide was incubated in a lysate or loaded into single cells although the fragments formed in significantly different ratios suggesting that distinct peptidases metabolized the peptide in the two preparations. The rate of peptide degradation and phosphorylation was on average 0.1 ± 0.2 zmol pg(-1) s(-1) and 0.04 ± 0.08 zmol pg(-1) s(-1), respectively, for single LNCaP cells loaded with 4 ± 8 μM of peptide. Peptidase-resistant kinase substrates should find widespread utility in both lysate-based and single-cell assays of kinase activity.     

Silver nanorod arrays as a surface-enhanced Raman scattering substrate for foodborne pathogenic bacteria detection

Surface-enhanced Raman scattering (SERS) using novel silver nanorod array substrates has been used for the detection of pathogenic bacteria. The substrate consists of a base layer of 500 nm silver film on a glass slide and a layer of silver nanorod array with a length of approximately 1 microm produced by the oblique angle deposition method at a vapor incident angle of 86 degrees . Spectra from whole cell bacteria, Generic Escherichia coli, E. coli O157:H7, E. coli DH 5alpha, Staphylococcus aureus, S. epidermidis, and Salmonella typhimurium, and bacteria mixtures have been obtained. This SERS active substrate can detect spectral differences between Gram types, different species, their mixture, and strains. Principal component analysis (PCA) has been applied to classify the spectra. Viable and nonviable cells have also been examined, and significantly reduced SERS responses were observed for nonviable cells. SERS detection of bacteria at the single cell level, excited at low incident laser power (12 micro W) and short collection time (10 s), has also been demonstrated. These results indicate that the SERS-active silver nanorod array substrate is a potential analytical sensor for rapid identification of microorganisms with a minimum of sample preparation.     

Differential phosphoprotein mapping in cancer cells using protein microarrays produced from 2-D liquid fractionation

A combination of protein microarrays and two-dimensional liquid-phase separation of proteins has been used for global profiling of the phosphoproteome in human breast cancer cells. This method has been applied to study changes in phosphorylation profile resulting from treatment of the cancer cells with PD173074, a known receptor tyrosine kinase inhibitor. The proteins separated by 2-D liquid-phase separation were arrayed on epoxy-coated glass slides and first screened for phosphorylation using fluorescent Pro-Q Diamond stain. The candidate proteins were then identified using MALDI/ESI MS/MS analysis. Further, validation was achieved by immunoblot analysis using anti-phosphotyrosine antibodies. A dynamic range of approximately 100 was achieved on the microarray when beta-casein was used as a standard protein for obtaining quantitative data. Importantly, the power of this method lies in its ability to identify a large group of proteins in a single experiment that are coregulated in their posttranslational modifications, upon treatment with the inhibitor. Since proteins are known to form interacting circuits that eventually lead to various signaling events, detection of such global phosphorylation profiles might enable delineation of functional pathways that play an important role during cancer initiation and progression.     

Peptide biosensors for the electrochemical measurement of protein kinase activity

The kinase activities are elucidated using the novel redox-active cosubstrate adenosine 5'-[gamma-ferrocene] triphosphate (Fc-ATP), which enables the kinase-catalyzed transfer of a redox active gamma-phosphate-Fc to a hydroxyamino acid. In this report, a versatile electrochemical biosensor is developed for monitoring the activity and inhibition of a serine/threonine kinase, casein kinase 2 (CK2), and protein tyrosine kinases, Abl1-T315I and HER2, in buffered solutions and in cell lysates. The method is based on the labeling of a specific phosphorylation event with Fc, followed by electrochemical detection. The electrochemical response obtained from the "ferrocenylated" peptides enables monitoring the activity of the kinase and its substrate, as well as the inhibition of small molecule inhibitors on protein phosphorylation. Kinetic information was extracted from the electrochemical measurements for the determination of K(m) and V(m) values, which were in agreement with those previously reported. Kinase reactions were also performed in the presence of well-defined inhibitors of CK2, 4,5,6,7-tetrabromo-2-azabenzimidazole, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, and E-3-(2,3,4,5-tetrabromophenyl)acrylic acid as well as the nonspecific kinase inhibitors, staurosporine and N-benzoylstaurosporine. On the basis of the dependency of the Fc signal on inhibitor concentration, K(i) of the inhibitors was estimated, which were also in agreement with the literature values. The performance of the biosensor was optimized including the kinase reaction, incubation with Fc-ATP, and the small molecule inhibitors. Peptide modified electrochemical biosensors are promising candidates for cost-effective in vitro kinase activity and inhibitor screening assays.     

Droplet-based microfluidic flow injection system with large-scale concentration gradient by a single nanoliter-scale injection for enzyme inhibition assay

We described a microfluidic chip-based system capable of generating droplet array with a large scale concentration gradient by coupling flow injection gradient technique with droplet-based microfluidics. Multiple modules including sample injection, sample dispersion, gradient generation, droplet formation, mixing of sample and reagents, and online reaction within the droplets were integrated into the microchip. In the system, nanoliter-scale sample solution was automatically injected into the chip under valveless flow injection analysis mode. The sample zone was first dispersed in the microchannel to form a concentration gradient along the axial direction of the microchannel and then segmented into a linear array of droplets by immiscible oil phase. With the segmentation and protection of the oil phase, the concentration gradient profile of the sample was preserved in the droplet array with high fidelity. With a single injection of 16 nL of sample solution, an array of droplets with concentration gradient spanning 3-4 orders of magnitude could be generated. The present system was applied in the enzyme inhibition assay of β-galactosidase to preliminarily demonstrate its potential in high throughput drug screening. With a single injection of 16 nL of inhibitor solution, more than 240 in-droplet enzyme inhibition reactions with different inhibitor concentrations could be performed with an analysis time of 2.5 min. Compared with multiwell plate-based screening systems, the inhibitor consumption was reduced 1000-fold.