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Cell-free protein expression in a microchannel array with passive pumping

Ruba Khnouf, a David J. Beebe* b and Z. Hugh Fan* ac

Received 12th May 2008, Accepted 1st September 2008

First published as an Advance Article on the web 22nd October 2008

DOI: 10.1039/b808034h

We report in vitro (cell-free) protein expression in a microfluidic device using passive pumping. The

polystyrene device contains 192 microchannels, each of which is connected to two wells positioned in

a 384-well microplate format. A larger droplet of an expression solution was placed at one well of each

channel while a smaller droplet of a nutrient solution was at the other well. Protein expression took

place in the larger droplet and we found the expression yield in the expression solution is enhanced due

to the replenishment of the nutrient solution supplied by passive pumping via the channel. The pumping

pressure was generated from the difference in the surface tension between two different sized droplets at

the two wells. We demonstrated expression of luciferase in the device and the expression yield was

measured using luminescence assay. Different experimental conditions were investigated to achieve

maximum protein yield with the least amount of reagents. Protein expression yields were found to be

dependent on the amount of the nutrient solution pumped, independent of the amount of the

expression solution within the experimental conditions studied. A higher feeding frequency or delivery

rate of the nutrient solution resulted in higher protein expression yield. The work demonstrated the

feasibility of using the microchannel array for protein expression with the following advantages: (1)

simultaneous production of the same protein with different conditions to optimize the expression

process; (2) simultaneous production of different proteins for high-throughput protein expression with

high yield; (3) low reagent cost due to the fact that it consumes 125–800 times less than the amount used

in a protein expression instrument commercially available.

Introduction

Proteins are important to life, since they regulate most molecular

and cellular functions. As a result, it is often necessary to produce

proteins for studying their molecular structures and functions. A

variety of protein expression (biological synthesis) systems have

been developed to synthesize proteins, including E. coli, yeast,

insect, and cell-free protein expression systems. 1

The cell-free protein expression method is appealing for high

throughput protein production, because it has an ability to

generate milligram quantities of proteins in hours and produce

proteins that cannot be expressed in vivo due to their toxic effect

on the physiology of the cell. 2 Proteins expressed in vitro have

been shown to have the same structures and characteristics as

those produced in vivo, making cell-free protein expression

a viable technique. 1 In addition, cell-free protein expression

simplifies protein purification and allows expression of multiple

functional proteins in parallel.

Different configurations have been investigated to maximize

the amount of proteins produced in cell-free protein expression

systems. 2–10 One is the batch process, in which the protein

synthesis machinery and other reagents are mixed together in one

a

Department of Biomedical Engineering, University of Florida, P.O. Box

116131, Gainesville, FL, 32611, USA

b

Department of Biomedical Engineering, University of Wisconsin-Madison,

Madison, WI, 53706, USA

c

Department of Mechanical and Aerospace Engineering, Interdisciplinary

Microsystems Group, University of Florida, P.O. Box 116250,

Gainesville, FL, 32611, USA. E-mail: hfan@ufl.edu; Fax: +1-352-392-

7303

container. The second one is the continuous flow configuration,

in which nutrients (e.g., amino acids and adenosine triphosphate

(ATP)) are continuously pumped to the expression container

while byproducts (e.g. hydrolysis products of triphosphates) are

removed with the pumped fluids. 3 This approach leads to higher

protein expression yield than the batch process because protein

synthesis will not terminate earlier due to fast depletion of energy

sources (ATP) and the removal of the byproducts eliminates

possible inhibition of the biochemical reactions. Continuous

supply of the nutrients for high-yield protein expression is similar

to supplying nutrients to biological cells for their long-term

viability. An alternative to the continuous flow configuration is

continuous-exchange cell-free (CECF) protein expression, in

which the addition of nutrients and the removal of byproducts

are achieved by exchanging the solutions in two chambers

separated by a dialysis membrane. 5

Protein expression using the CECF configuration has also

been implemented in microfluidic devices and miniaturized

chambers. 11–13 A dialysis membrane was used to separate reaction

and nutrient chambers fabricated in poly(methyl methacrylate)

11,12 or polydimethylsiloxane. 13 A bilayer to separate

transcription and translation reagents has also been studied in

a microplate. 14 Microreactors have also been fabricated in

a silicon wafer to carry out cell-free protein expression. 15,16

Water-in-oil emulsion is another approach to achieve compartmentalization

of two solutions for in vitro protein synthesis. 17,18

In this work, we implemented cell-free protein expression in

a microchannel array that has characteristics of continuous flow

and CECF configurations. The ports of the device are in a format

of a 384-well microplate with 192 microfluidic channels

56 | Lab Chip, 2009, 9, 56–61 This journal is ª The Royal Society of Chemistry 2009


connecting pairs of ports. When a larger droplet of the protein

expression solution is placed at the outlet of a channel and

a smaller droplet of the nutrient solution at the inlet, passive

pumping occurs driving the fluid from the inlet through the

channel to the outlet because of the difference in the size of these

two droplets and their surface tensions. 19–21 This pumping

mechanism allows for continuous supply of nutrients from the

inlet to the outlet where protein expression takes place. 22 The

byproducts are also diluted, reducing possible inhibitory effects.

The array format allows simultaneous production of the same

protein with different conditions, facilitating screening for the

optimum expression conditions. It also enables simultaneous

production of different proteins for high-throughput protein

production, matching high-throughput gene discovery. We

utilized the device for the expression of luciferase and studied the

effects of different experimental conditions on the protein

expression yield. The work demonstrated the feasibility of using

the microchannel array for high-throughput protein expression

with high expression yield. More than 2 orders of magnitude of

savings in the reagent cost can be achieved since the microchannel

array consumes 125–800 times less than the amount used

in a protein expression instrument commercially available.

Experimental

Reagents and materials

RTS 100 wheat germ kits were purchased from Roche (Mannheim,

Germany). Each kit consists of wheat germ lysis buffer,

reaction mix, feeding mix, amino acids, and methionine. T7

luciferase DNA vector, luciferase assay reagent, and nuclease

free water were obtained from Promega Corporation (Madison,

WI, USA). Purified water for rinsing devices was obtained from

a Barnstead Nanopure water system (Dubuque, IA, USA).

Device fabrication

The microchannel arrays were fabricated by Edge Embossing,

LLC (Medford, MA, USA) using the following process flow. The

desired microstructures were created in an acrylic master using

CNC milling. Next, a soft elastomer tool was cast from the

acrylic master and resin pellets of polystyrene (PS 168 N,

Frantschach Rothrist AG) were placed in the soft tool cavity. A

combination of temperature and pressure were applied to thermoform

the resin. The embossed polystyrene part was them

exposed to a 20% solution of dicholormethane for 1 min, rinsed

in ethanol, and then placed in contact with a Nunc OmniTray. A

second temperature and pressure cycle was then performed on

the assembly to induce solvent-assisted thermal bonding.

Protein expression

Luciferase was produced using a RTS 100 kit. A protein

expression solution and a feeding (nutrient) solution were

prepared by following the protocols recommended by the

manufacturer. The protein expression solution consists of 15 mL

wheat germ lysis buffer, 15 mL reaction mix, 4 mL amino acids,

1 mL methionine, 2 mL T7 luciferase DNA vector, and 13 mL

nuclease free water. The nutrient solution contains 900 mL

feeding mix, 80 mL amino acids, and 20 mL methionine. The

Fig. 1 (a) Picture of the devices used in this work. The device consisted

of 192 channels and 384 wells. Each pair of two adjacent wells was

connected by one channel. (b) For each channel, a larger droplet was

placed in the outlet, followed by a smaller droplet in the inlet. The

difference in the surface tension between two different sized droplets

generated a pumping pressure, producing a flow in the channel from the

inlet to the outlet. (c) Protein expression in the droplet at the outlet

consisted of DNA transcription and protein translation.

production of luciferase using this kit has been implemented in

conventional containers or devices as described previously. 11,12

To implement protein expression in the passive pumping

device (Fig. 1), a channel was first rinsed once with purified

water. Protein expression solution (4–8 mL) was then pipetted

into the outlet of the channel; the solution flowed through the

channel towards the inlet due to pipetting and the capillary force.

The inlet was then filled with an appropriate amount of the

nutrient solution using a pipette. Note that the amount of the

nutrient solution at the inlet was always less than the amount of

the expression solution at the outlet so that passive pumping

took place.

In addition to using a pipette, we also employed a syringe

pump for experiments that required a continuous supply of the

nutrient solution. A syringe pump (UltraMicropump II) from

World Precision Instruments Inc. (Sarasota, Fl) was connected

to the solution droplet at the inlet via a syringe needle. Note

that the syringe pump is for continuous delivery of the nutrients,

not for pumping it through the channel. The delivery speed

(33–1000) nL/s was set to maintain the droplet size at the inlet

smaller than that at the outlet.

At the end of every experiment, the droplet of the expression

solution at the outlet (as well as most solution in the channel) was

collected using a pipette. There was no droplet of the nutrient

solution at the inlet at the end of every experiment since the inlet

droplet disappeared within a couple of seconds (due to passive

pumping to the outlet). The amount collected depended on the

amount of the initial expression solution, the amount of nutrient

solution pumped, and the duration of the experiment. A fixed

amount (5 mL) of the collected sample was used to assay the

concentration of luciferase expressed in each experiment.

The assay was performed using a Mithras microplate reader

(Berthold Technologies, Germany). Each of the collected

samples (5 mL) was added to a well in a standard 384-well

microplate. The plate was then placed in the reader, which was

programmed to inject 35 mL of luciferase assay reagent, followed

by a 2 s shake. The luminescence signal of the following 10 s was

measured and reported by the reader, and it is correlated to the

amount of luciferase.

This journal is ª The Royal Society of Chemistry 2009 Lab Chip, 2009, 9, 56–61 | 57


Results and discussion

Device design and protein expression

A picture of the device is shown in Fig. 1a, in which there are 192

channels and 384 wells. Each pair of two adjacent wells is connected

by one channel. The distance between the well centers is

4.5 mm, following the microplate standards defined by the

Society for Biomolecular Screening (SBS) and accepted by the

American National Standards Institute.

As indicated in Fig. 1b, one well of each channel is designated

as an inlet while the other well is an outlet. A larger droplet is first

placed in the outlet, followed by a smaller droplet in the inlet. As

explained previously, 19 the difference in the size of the two

droplets at two wells generated a pumping pressure because of

the surface tension in their curved surfaces. As a result, a flow

was produced in the channel from the inlet to the outlet, as

illustrated in Fig. 1b. Note that the flow was continuous; it

stopped when the inlet droplet shrank to a slightly curved shape

that was at equilibrium with the outlet droplet. 19

When the protein expression solution was placed in the outlet,

protein synthesis took place as shown in Fig. 1c. Protein expression

consisted of two steps: a DNA template consisting of

a coding sequence is transcribed into a messenger RNA; and the

RNA is then translated into the corresponding protein. The

transcription and translation steps were coupled together

and occurred in the same reaction mixture. When the nutrient

solution was added into the inlet either intermittently or continuously,

nutrients were supplied through the channel to the outlet.

Continuous supply of the nutrients enabled the protein expression

to continue for a long period of time, in an analogy to supplying

nutrients to biological cells for their long-term viability.

Fig. 2 shows the luciferase expression yield in the passive

pumping device. The yield is indicated by the luminescence signal

in relative light units (RLU) per second from the microplate

reader when 5 mL of the collected droplet at the outlet was

assayed using luciferase assay reagent as described in the

Experimental section. The error bars were obtained from four

repeat experiments in different channels in the same device,

indicating the channel-to-channel variation as well as the

inherent protein expression variation. In these experiments, 6 mL

of the protein expression solution was initially pipetted into the

Fig. 2 Comparison in luciferase expression yield between a microcentrifuge

tube and the passive pumping device. The yield is represented

by the luminescence signal of the luciferase assay while the error bars

indicate the standard deviation of signals from four repeat experiments in

different channels.

outlet, followed by filling 4 mL of the nutrient solution. After

passive pumping of the nutrient solution from the inlet to the

outlet and 10 min of reactions, another 4 mL of the nutrient

solution was added into the inlet using a pipette; this step was

repeated every 10 min. A total of 12 mL of the nutrient solution

was added into the inlet while the whole experiment lasted for

30 min. Note that the droplet size of the nutrient solution at the

inlet was always smaller than the droplet size of the expression

solution at the outlet so that the passive pumping took place.

Both droplets were exposed to air, so that evaporation occurred

at the same time. However, evaporation was contained to a large

degree since the device was covered with a lid that came with the

device.

For comparison, we did the same experiments in a microcentrifuge

tube. The same amount (6 mL) of the protein expression

solution was used; and the reaction was allowed to last for

the same period of time (30 min). And 5 mL of the expression

products was assayed in the same way. As shown in Fig. 2, the

expression yield in the microcentrifuge tube was 4.9 times lower

than in the device. The results suggest that the additional nutrients

supplied by passive pumping in the device prolonged the

biochemical reactions and resulted in higher protein expression

yield. Note that simply mixing 6 mL of the expression solution

with 12 mL of the nutrient solution in a tube led to a 4.1 times

increase in expression yield (compared to the same amount of the

expression solution in a tube without the nutrient solution).

Incorporation of a dialysis membrane in a well-in-well device

described previously 11 could result in a 121 time increase in

expression yield, though a much larger amount of nutrient

solution (200 mL) and a larger amount of expression mix (10 mL)

were used in that case.

Nutrient solution amount

We then quantitatively studied the effects of the amount of

nutrient solution on the protein expression yield. The protein

expression solution initially pipetted into the outlet remained the

same at 6 mL. However, the amount of nutrient solution initially

added into the inlet and added consecutively afterwards was 1, 2,

3, or 4 mL at a frequency of once every 10 min (i.e., a total of 24

mL was added in one hour for the 4 mL treatment). They were

added into the inlet using a pipette. Each experiment lasted for

one hour; the same reaction time was used for all experiments.

Fig. 3 shows the protein expression yield as a function of the

amount of the nutrient solution. The result indicates that the

protein expression yield exponentially increased with the amount

of the nutrient solution in the range of the experimental conditions

we studied. The larger the amount of the nutrient solution,

the greater the quantity of the nutrients for protein expression,

and the higher the protein expression yield.

It is worth noting that the amount of the nutrient solution

could not be significantly larger than 4 mL without causing the

droplet at the outlet to deform, disrupting the passive pumping

mechanism. In addition, too large amount of the nutrient solution

could lead to a larger droplet at the inlet than at the outlet,

causing the fluids to move in the opposite direction, i.e. from the

outlet to the inlet through the same passive pumping mechanism.

To address the concern of adding too much solution at once

and to maximize the effect of the nutrient solution amount, we

58 | Lab Chip, 2009, 9, 56–61 This journal is ª The Royal Society of Chemistry 2009


Fig. 3 Effects of the amount of the nutrient solution on protein

expression yield. The nutrient solution was added using a pipette. The x

axis indicates the amount of the nutrient solution added every 10 min.

The total reaction time was one hour. The line is the best fit of the

exponential regression.

examined using a syringe pump to add the nutrient solution

continuously. A syringe pump was connected to the inlet droplet

via a needle. Note that the syringe pump is not used for pumping

fluids in the channel, rather for delivering reagents into the inlet

droplet. In other words, the needle was placed in contact with the

inlet droplet in open air. A delivery speed of 1000 nL/s was

chosen to maintain the passive pumping between two droplets.

The delivery duration determined the amount of the nutrient

solution added. Protein expression reactions were allowed for

30 min after the initiation of delivery, and the samples were

collected and measured as described above. Fig. 4a shows the

protein expression yield as a function of the amount of nutrient

solution. A trend similar to our previous account (Fig. 3) was

initially observed that the protein expression yield increased with

the amount of nutrient solution. However, the yield reached

a plateau at 12 mL of nutrient solution and then fell off at 20 mL.

To investigate if the trend observed is a result of the device, we

studied the same experiments in microcentrifuge tubes. Different

amounts of the nutrient solution were added to the same amount

(6 mL) of the expression solution. The mixture was also allowed

to react for 30 min and the luminescence was then measured.

Fig. 4b shows a similar trend as in Fig. 4a, suggesting that the

decrease in the protein expression yield when too much nutrient

solution was used is a characteristic of the reactions, rather than

the device.

The phenomenon can be explained by the increased dilution of

the protein synthesis machinery when too much nutrient solution

was added. Keeping the reactions for a long period of time while

allowing the additional nutrient solution to diffuse slowly into

the expression mixture will not significantly dilute the expression

synthesis machinery at any moment, resulting in higher protein

expression yield. 3 However, too much nutrient solutions at once

will dilute the expression synthesis machinery, likely slowing the

reaction kinetics. This explains the difference between Fig. 3 (the

nutrient solution supplied over time) and Fig. 4 (when the syringe

pump was supplying the nutrient solution too fast).

Another plausible explanation is related to the different roles

played by catalysts, for example, magnesium ions (Mg 2+ ). It has

been reported that the amount of Mg 2+ required for transcription

(DNA to mRNA) is higher than for translation (mRNA to

proteins) in the wheat germ expression system. 6 As a result, the

Mg 2+ concentration in the expression solution is higher than in

the nutrient solution in the kits, reflecting the fact that transcription

takes place prior to translation. Adding too much

nutrient solution to the expression solution reduces the concentration

of Mg 2+ , resulting in the reduction in the transcription

yield and accordingly the overall expression yield. It is possible to

solve the issue by adding an extra amount of Mg 2+ to the

nutrient, but it will likely have adverse effects on translation.

These discussions signify the advantage of using the device to

carry out protein expression because it allows the additional

reactants to pump slowly into the reaction mixture, keeps the

reactions for a longer period of time, and leads to higher protein

expression yield. 23

Feeding frequency and delivery rate

Fig. 4 Effects of the amount of nutrient solution on protein expression

yield. The nutrient solution was added using a syringe pump. The reaction

time of each experiment was 30 min. Experiments were carried out in

either the passive pumping devices (a) or in a microcentrifuge tube (b).

Since the amount of the nutrient solution has an effect on the

protein expression yield, we studied the feeding frequency (i.e.,

the number of adding the nutrient solution per unit time) on the

protein expression yield. In all experiments, the total amount of

nutrient solution is the same over the same period of time.

However, the amount added each time and the time interval

between feedings were different. Fig. 5a shows the protein

expression yield as a function of the feeding frequency. The

studied feeding frequencies are 0.0067 Hz (once every 2.5 min),

0.0033 Hz (once every 5 min), and 0.0017 Hz (once every 10 min).

The nutrient amounts are 1 mL, 2 mL, and 4 mL, respectively. As

a result, the total amount of the nutrient solution added was

24 mL in one hour for all three cases. The results in Fig. 5a

suggest that protein expression yield is higher at a higher feeding

frequency while there is no significant difference between low

frequency feedings.

The increase in the expression yield at a higher feeding

frequency is likely a result of increased mixing when the nutrient

This journal is ª The Royal Society of Chemistry 2009 Lab Chip, 2009, 9, 56–61 | 59


Fig. 6 Effects of the amount of the expression solution on protein

production. The same amount (12 mL) of the nutrient solution was added

using a syringe pump with a delivery rate of 1000 nL/s while the amount

of the expression solution varied as indicated.

Fig. 5 (a) Effects of the feeding frequency of the nutrient solution on

protein production. A total volume of 24 mL of the nutrient solution was

added by pipetting 1, 2, and 4 mL at a time interval of 2.5, 5, and 10 min,

respectively, over a period of one hour. (b) Effects of the delivery rate of

the nutrient solution on protein production when a syringe pump was

used. A total volume of 12 mL of the nutrient solution was delivered into

the inlets of different channels of the device using various delivery rates.

solution is frequently supplied. 22 At a higher feeding frequency,

a smaller amount of nutrient solution was supplied in a shorter

time interval, resulting in a smaller droplet at the inlet that

caused an increase in the pumping pressure and the flow velocity

according to Berthier and Beebe. 22 A higher flow velocity

enhanced the degree of mixing. More mixing is known to

improve the reaction kinetics and increase cell-free protein

expression yield. 24

To confirm the effect of the feeding frequency, we also investigated

using the syringe pump as the feeding approach. The

delivery rates of the nutrient solution ranged from 33 nL/s to

1000 nL/s, and the delivery time varied to maintain that the total

nutrient solution pumped was 12 mL. Protein expression reactions

were allowed for 30 min after the initiation of delivery.

Fig. 5b shows the protein expression yield as a function of the

delivery rate. The result indicates that protein production yield

increased initially with the delivery rate of the nutrient solution.

Similarly, the observation can be explained by the increase in

mixing at a higher delivery rate.

Expression solution amount

Since one of the goals of this research is to achieve the maximum

protein production with the least amount of reagents and cost,

we studied the effects of the amount of the expression solution on

protein production. Different amounts of the expression solution

were placed in the outlets while the same amount of the nutrient

solution was used in the inlets. The nutrient solution (12 mL) was

added using a syringe pump with a delivery rate of 1000 nL/s.

Fig. 6 shows the protein expression yield as a function of the

amount of the expression solution. The results indicate there is

no significant difference among experiments when different

amounts of expression solution were used, at least within the

range of the experimental conditions we used. Statistical analysis

using ANOVA (analysis of variance) 25 indicates that the luminescence

signals in Fig. 6 are the same at the 95% confidence level

for different amounts of the expression solution. The results are

significant since we can use a smaller amount of the expression

reagents for protein production as long as enough amount of the

nutrient solution is used.

It should be useful to compare the amount of the reagents used

in this device with those required in a commercial CECF machine

called Rapid Translation Systems (RTS). 26 In this work, we used

5–8 mL of the expression solution; this volume is 125–200 times

less than 1 mL of an expression solution in RTS 500. Similarly,

the volume of the nutrient solution is 12–24 mL, which is 400–

800 times less than 10 mL of a feeding solution in RTS 500. The

decrease in the volume of both expression solution and nutrient

solution will significantly reduce reagent consumption when

using cell-free protein expression for high-throughput assays. 27

As a result, the device will be extremely useful in situations where

a large number of proteins need to be screened and the amount of

proteins produced in the device is sufficient for assays.

Comparison of the results in Fig. 6 with that in Fig. 3 indicates

that the amount of nutrient solution has a greater effect on the

protein production than the amount of expression solution. This

finding is in agreement with incorporation of microfluidic

channels for supplying nutrients, as well as in agreement with the

reports using continuous flow or CECF configurations. 3,5

Conclusion

Production of luciferase using a cell-free expression system was

demonstrated in a microchannel array device. Cell-free protein

expression systems address the challenges encountered in cellbased

methods, including reagent and facility cost, time and

labor consumption, and difficulty in separating proteins of

interest from other cellular proteins. This protein production

method will be useful in proteomics studies, drug discovery, and

fundamental scientific and biomedical research. 23,24,27

Passive pumping in a microchannel is a method meeting the

requirements of cell-free protein expression. The nutrient

60 | Lab Chip, 2009, 9, 56–61 This journal is ª The Royal Society of Chemistry 2009


solution in a smaller droplet is passively pumped through

a channel to the protein expression solution in a larger droplet,

achieving continuous replenishment of nutrients. As a result,

protein expression yield is enhanced by 5 times. Using luciferase

expression as an example, we studied the effects of different

experimental conditions on the protein expression yield. We

found that the expression yields are dependent on the amount of

nutrient solution supplied, and independent of the amount of

expression solution within the experimental conditions studied.

A higher feeding frequency or a higher delivery rate of the

nutrient solution resulted in higher protein expression yield.

These results will help achieve maximum protein yield. In addition,

we showed more than 2 orders of magnitude of savings in

the reagent consumption than a protein expression instrument

commercially available.

The protein expression device is in a 384-well microplate

format, which is compatible with commercially available reagent

dispensers and microplate readers. As a result, the method and

device are adaptable to high-throughput protein production. In

addition, we demonstrated an acceptable repeatability among

the experiments in different channels. Therefore, the device may

provide a way to achieve protein expression in parallel, with high

yield and low cost.

Acknowledgements

This work is supported by Defense Advanced Research Projects

Agency (DARPA) via Micro/Nano Fluidics Fundamentals

Focus Center at the University of California at Irvine.

References

1 P. Braun and J. LaBaer, Trends Biotechnol., 2003, 21, 383–388.

2 Y. Endo and T. Sawasaki, Curr. Opin. Biotechnol., 2006, 17, 373–

380.

3 A. S. Spirin, V. I. Baranov, L. A. Ryabova, S. Y. Ovodov and

Y. B. Alakhov, Science, 1988, 242, 1162–1164.

4 F. Katzen, G. Chang and W. Kudlicki, Trends Biotechnol., 2005, 23,

150–156.

5 A. S. Spirin, Trends Biotechnol., 2004, 22, 538–545.

6 T. W. Kim, D. M. Kim and C. Y. Choi, J. Biotechnol., 2006, 124, 373–

380.

7 T. Kigawa, T. Yabuki, Y. Yoshida, M. Tsutsui, Y. Ito, T. Shibata and

S. Yokoyama, Febs Lett., 1999, 442, 15–19.

8 M. C. Jewett and J. R. Swartz, Biotechnol. Prog., 2004, 20, 102–109.

9 Y. Shimizu, A. Inoue, Y. Tomari, T. Suzuki, T. Yokogawa,

K. Nishikawa and T. Ueda, Nat. Biotechnol., 2001, 19, 751–755.

10 M. A. Coleman, V. H. Lao, B. W. Segelke and P. T. Beernink,

J. Proteome Res., 2004, 3, 1024–1032.

11 Q. Mei, C. K. Fredrickson, W. Lian, S. Jin and Z. H. Fan, Anal.

Chem., 2006, 78, 7659–7664.

12 Q. Mei, C. K. Fredrickson, A. Simon, R. Khnouf and Z. H. Fan,

Biotechnol. Prog., 2007, 23, 1305–1311.

13 G. H. Hahn, A. Asthana, D. M. Kim and D. P. Kim, Anal. Biochem.,

2007, 365, 280–282.

14 T. Sawasaki, Y. Hasegawa, M. Tsuchimochi, N. Kamura,

T. Ogasawara, T. Kuroita and Y. Endo, FEBS Lett., 2002, 514,

102–105.

15 T. Nojima, T. Fujii, K. Hosokawa, A. Yotsumoto, S. Shoji and

I. Endo, Bioprocess Eng., 2000, 22, 13–17.

16 K. Hosokawa, T. Fujii, T. Nojima, S. Shoji, A. Yotsumoto and

I. Endo, J. Micromechatronics, 2000, 1, 85–98.

17 A. Rothe, R. N. Surjadi and B. E. Power, Trends Biotechnol., 2006,

24, 587–592.

18 P. S. Dittrich, M. Jahnz and P. Schwille, Chembiochem, 2005, 6, 811–

814.

19 G. M. Walker and D. J. Beebe, Lab Chip, 2002, 2, 131–134.

20 J. Warrick, I. Meyvantsson, J. Ju and D. J. Beebe, Lab Chip, 2007, 7,

316–321.

21 I. Meyvantsson, J. W. Warrick, S. Hayes, A. Skoien and D. J. Beebe,

Lab Chip, 2008, 8, 717–724.

22 E. Berthier and D. J. Beebe, Lab Chip, 2007, 7, 1475–1478.

23 L. Falzon, M. Suzuki and M. Inouye, Curr. Opin. Biotechnol., 2006,

17, 347–352.

24 K. Ozawa, M. J. Headlam, D. Mouradov, S. J. Watt, J. L. Beck,

K. J. Rodgers, R. T. Dean, T. Huber, G. Otting and N. E. Dixon,

Febs J., 2005, 272, 3162–3171.

25 R. L. Anderson, Practical statistics for analytical chemists, Van

Nostrand Reinhold, New York, 1987.

26 J. M. Betton, Curr. Protein Pept. Sci., 2003, 4, 73–80.

27 P. Angenendt, L. Nyarsik, W. Szaflarski, J. Glokler, K. H. Nierhaus,

H. Lehrach, D. J. Cahill and A. Lueking, Anal. Chem., 2004, 76,

1844–1849.

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