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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Am Chem Soc. Author manuscript; available in PMC Aug 5, 2010.
Published in final edited form as:
PMCID: PMC2739094
NIHMSID: NIHMS131740
Microfluidic concentration-enhanced cellular kinase activity assay
Jeong Hoon Lee,1# Benjamin D. Cosgrove,2@ Douglas A. Lauffenburger,2 and Jongyoon Han1,2*
1 Department of Electrical Engineering and Computer Science, 77 Massachusetts Avenue, Cambridge, MA 02139
2 Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139
jyhan/at/mit.edu
#Current Address: Department of Electrical Engineering, Kwangwoon University, Seoul, Korea
@Current Address: Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, CA.
Abstract
In this paper, we reported a simple, disposable PDMS micro/nanofluidic preconcentration chip for In-vitro concentration-enhanced cell kinase assays. Utilizing the preconcentration (electrokinetic trapping) directly from cell lysate (1 mM ATP) samples, we could achieve at least 25-fold increase in reaction velocity and 65-fold enhancement in sensitivity. In addition, we shorten the assay time down to less than 10 mins, with the sample volume requirements of down to ~5 cells. This device could be a generic and powerful tool for diagnostics and systems biology studies at the single-cell level, if properly optimized and integrated with the cell culture microdevices.
In cell signaling pathways, information is transmitted via specific protein-substrate binding interactions, typically leading to modification of the substrate modulating a next biochemical or biophysical step in the signaling cascade. Ability to experimentally monitor the enzymatic activity of each kinase within multi-pathway signaling networks is critical for understanding complicated network dynamics. Many technologies are available for measuring and monitoring the signaling processes in regulatory pathways.1 For affinity-based technologies (kinase assays), availability of specific antibodies (or equivalent) toward the given target has always been the main hurdle for general application of the technique. Also reaction times required to turn over enough substrates for detection can be long, typically a few hours. While mass spectrometry (MS) is a versatile measurement tool for proteins with the ability of discerning even subtle post-translational modifications, throughput is generally low, with the requirements of relatively large sample size (105~107 cells). As a result, measurement of signals within regulatory pathways is largely done by averaging over many cells, which may or may not be at the same stages of intracellular signal processing. It is widely accepted, therefore, that such a population-average measurement provides only a ‘blurred’ picture of inner workings of the complex pathways.24 Although there are few reports of measurement technologies capable of assaying kinase activities in single cells, these rely on injection of specialized kinase substrates directly into living cells5 or require imaging6 or flow cytometry7 approaches and thus are not compatible with other types of assays. Here, we describe a novel microfluidics approach for assaying kinase activity in samples from small numbers of cells using standard cell culture and lysis methods compatible with other types of lysate-based assays (e.g. real-time polymerase chain reaction) and integrateable into existing lab-on-a-chip approaches.
Various types of enzyme activity assays have been implemented in microfluidic chips previously810. However measurement of low abundance kinase activities has not yet been reported, mainly due to the low kinase turnover rate. Recently, we have developed a novel nanofluidic biomolecule concentration device which can be used to collect and trap proteins contained in a given sample into a much smaller volume, thereby increasing the local concentration significantly.11,12 Using the concentration-enhancing device, we also reported a method of increasing both kinetics and sensitivity of immunoassays13, as well as the trace level enzyme (trypsin) activity assay14. So far these assays have been demonstrated only in a standard buffer system, not in a complex, real physiological sample as required for routine applications.
Herein, we report concentration-enhanced cell kinase assays in micro/nanofluidic platform directly from cell lysates, in order to enable scientific studies on cell signaling pathways at the single cell level. We demonstrate the concentration-enhanced enzyme assay with two important cellular kinases, MK2 and PKA, from human HepG2 cells (table S1) only with lysates from a few cells.
The key operations as well as the poly(dimethylsiloxane) (PDMS) nanofluidic preconcentrator chip are shown in Fig. 1. The micro/nanofluidic preconcentration chip was composed of two microchannels with a planar ion-selective Nafion membrane® across the microchannels12. The middle channel is where the ion depletion zone is created, which is used to trap proteins contained within cell culture lysate from HepG2 cells, as well as a kinase-activated fluorogenic chemosensor (Sox substrate15). The outer channel, which surrounds the sample channel on the left and right in the middle section of the device (Fig. 1a, also see Figure S1), is also filled with a 1X PBS buffer solution. By using a Nafion nanojunction that shows a high ionic permselectivity12 we can achieve stable concentration operations even in a complex physiological samples (table S1) with relatively high ionic strength. Detailed experimental procedures can be found in the Supporting Information (Figure S1 to S3).
Figure 1
Figure 1
Schematic representation of the concentration-enhanced cell kinase assays using micro/nanofluidic preconcentration chip. (a) Outline Schemes with off-chip preparation and on-chip kinase assay (b) The operation mode. (c) Assay schemes of PDMS preconcentration (more ...)
HepG2 cell lysate was prepared to have an overall protein concentration of ~125μg/ml. The cells used in this experiment have about 1ng of protein per cell, so this lysate represents ~125 cells/μl. We diluted 0.5μl of such lysates into 4.5μl of the premixed biosensor cocktail (with ATP, inhibitor cocktails, kinase buffer and 10mM Sox substrates). The resulting 5 μl lysate-Sox substrate mix (which contains cellular proteins from roughly 65 cells) was then loaded into the sample reservoir of the device. (Sample 1/1′ and 2/2′ in Fig. 2) Also as a positive control, recombinant enzyme (MK2 (125ng/ml) and PKA (60ng/ml)) was mixed in 1X PBS buffer and used in the assay (Sample 3/3′ in Fig. 2). Then, reaction to turn over Sox substrates (to render them fluorescent) by kinases were monitored, both with (conditions 1′~3′) and without (conditions 1~3) concentration enhancement. All the kinase activities were checked with the reaction in the standard microplate reader before the assays in microfluidic chip (Figure S4).
Figure 2
Figure 2
Performance of concentration-enhanced kinase activity assay (a) Fluorescence enhancement of untreated HepG2 lysates (1), NaCl-stimulated HepG2 lysates (2), and recombinant active MK2 enzyme (3) in a MK2 kinase assay with concentration-enhancement using (more ...)
We observed no fluorescence signal changes (with or without concentration enhancement) for our negative control samples (contains lysate buffer but not HepG2 cell lysate). Fluorescence intensities of turned-over substrates increase linearly with assay time. It was shown, both for MK2 and PKA tested, that the sensitivity of the activity measurement has been improved significantly due to the concentration enhancement. For example, comparison between condition 2′ and 2 in Fig. 2a shows ~11-fold increase in turnover rate (rate of increase in signal) of MK2. This allowed us to clearly differentiate the conditions 1′(1) and 2′(2) within 7 mins (see Figure S5), which was not possible in the unconcentrated assay, where traces for conditions 1 and 2 are shown to overlap even after an hour of reaction time (Figure 2a). In the experiment with PKA (Fig. 2b), similar enhancement was observed. Without concentration enhancement, no PKA signal was observed in untreated HepG2 lysates, Forskolin-treated HepG2 lysates, and recombinant PKA even after 60 mins. But, with the concentration enhancement, the Sox substrate phosphorylation by PKA was significantly increased. The activated HepG2 lysate sample (with 30-min Forskolin treatment, Fig. 2b, data 2′) demonstrated a ~11-fold increase in fluorogenic substrate phosphorylation, compared with the non-concentrated assay (Fig. 2b, data 2).
We repeated the same experiment with diluted cell lysates (down to 1.9μg/ml) in order to test the ultimate sensitivity of the assay. Table 1 shows MK2 kinase reaction velocity as a function of lysate or kinase concentration; these results demonstrate that the reaction rate and the sensitivity of low-concentration kinase activity measurement can be dramatically increased by implementing a 20-min concentration. Without concentration, the MK2 kinase reaction velocity at 125μg/ml lysate concentration (125ng/ml for recombinant kinase) was 0.045, 0.052 and 0.185 AU/min for untreated lysates, NaCl-treated lysates, and recombinant MK2, respectively. With concentration, these values were increased to 0.49, 1.43 and 5.16 AU/min, respectively, which is a ~25-fold increase in reaction velocity. Furthermore, with concentration, NaCl-treated MK2 cell lysates can be measured down to a concentration of 1.9μg/ml, which is at least a 65-fold enhancement in limit of detection. Detection from ~10μg/ml lysate, which is a conservative estimate of our current detection limit, represents kinase assay from ~5 cells. This clearly demonstrates the applicability of our assay for single cell level study of cell regulatory pathways.
Table 1
Table 1
MK2-Sox reaction velocity as a function of cell lysate (recombinant kinase) concentration (AU/min)
The sensitivity enhancement was more prominent in the detection of diluted lysates. We suspect that this is caused by the additional interference from inhibiting enzymes in higher-concentration cell lysate samples. If inhibiting enzymes are concentrated along with the target kinases above a certain level in the concentration plug, they can (non-specifically) interact with the target kinases, reducing their turn-over rate. Presumably, this would be less of a problem in detections using lower-cell number samples.
In conclusion, we have demonstrated a novel device for in vitro concentration-enhanced cell kinase assays, with at least 25-fold increase in reaction velocity and 65-fold enhancement in sensitivity. In addition, we shorten the assay time from ~1 hour to 10–20 mins, as well as decreasing the amount of sample needed down to ~5μL (from ~200 μL using standard methods). This device, with its simplicity and efficient capability for assaying kinase activity in physiologically complex and relevant samples, could be a generic and powerful tool for diagnostics and systems biology research. If optimized, this scheme could lead to the quantitative measurement of cellular kinase activities potentially at or near single-cell level, which would provide an unblurred picture of inner-workings of cell regulatory pathways.
Supplementary Material
1_si_001
Acknowledgments
This work was supported by NIH grants R01-EB005743, R01-CA119402, P50-GM68672, as well as DoD Army Institute for Collaborative Biotechnologies.
Footnotes
Supporting Information Available: Detailed experimental procedure, cell kinase activity data using microplate reader, and the complete Ref (4) citation. This information is available free of charge via the Internet at http://pubs.acs.org/.
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