1
“Smart” mobile affinity matrix for microfluidic immunoassays

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj1

2
There is a current need for simple methods for immobilizing biomolecules within microfluidic channels.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot1

3
Here, a technique is reported for reversibly immobilizing immunoassay components in a channel zone that can be simply controlled by integrated heating elements.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

4
Latex beads were modified with the temperature-responsive polymer poly(N-isopropylacrylamide) (PNIPAAm) and co-modified with biotinylated poly(ethylene glycol) (PEG).

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

5
PNIPAAm undergoes a hydrophilic-to-hydrophobic transition when the temperature is raised above the lower critical solution temperature (LCST) (∼28 °C in the solutions used here).

Type: Method | Advantage: Yes | Novelty: New | ConceptID: Met1

6
This reversible transition drives the aggregation and dis-aggregation of the modified beads in heated zones within poly(ethylene terephthalate) (PET) microchannels.

Type: Method | Advantage: Yes | Novelty: New | ConceptID: Met1

7
Biotinylated monoclonal antibodies for the drug digoxin were bound via streptavidin to the biotin-PEG-coated beads.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

8
These antibody-functionalized beads were then reversibly immobilized by aggregation and hydrophobic adhesion to the surface of PET microfluidic channels in response to a thermal stimulus.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

9
The antibodies on the beads immobilized in the channel were shown to bind digoxin and a competitor fluorescent ligand from a flow stream in a quantitative competitive assay format that reported the digoxin concentration.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res1

10
The antibodies could be replenished for each immunoassay trial, using the reversible, temperature-controlled immobilization process.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res2

11
This technique allows reagent immobilization immediately prior to an analytical procedure, following the removal of previously utilized beads, guaranteeing fresh and active immobilized biomolecules.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con2

12
Furthermore, it provides a simple approach to multiplexing through the simultaneous or sequential injection of different antibody-coated bead species, potentially at multiple sites in the integrated device channels.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con3

Introduction

13
Many important bioanalytical techniques require an immobilized, biochemically active phase.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot2

14
Such techniques include affinity chromatography, microtiter plate-based immunoassays, genomic and proteomic microarray analysis,1,2 and micro/nanobead-based assays and separation processes.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met2

15
The translation of equivalent strategies to the microfluidic environment has relied on three broad categories of biomolecular immobilization techniques: surface modification of microfluidic channel walls,3-8 packing of microfluidic channels with biomolecule-bearing beads,9-14 and packing of microfluidic channels with biomolecule-bearing porous slabs.15,16

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met3

16
A disadvantage which most current microfluidic immobilization systems share is their inherent irreversibility.

Type: Method | Advantage: No | Novelty: Old | ConceptID: Met3

17
Once a channel is packed with beads or a monolithic polymer, or a channel surface has been chemically modified, there is no simple way for the device end user to remove or renew the immobilized molecule, or to add a new immobilized molecule.

Type: Method | Advantage: No | Novelty: Old | ConceptID: Met3

18
This limits the flexibility of device manufacturing, since each device must be manufactured with a specific immobilized biochemistry for a specific application.

Type: Method | Advantage: No | Novelty: Old | ConceptID: Met3

19
It also limits device lifetime, both on the shelf and in use, since biomolecules are often fragile, and non-renewable immobilized molecules will eventually lose activity.

Type: Method | Advantage: No | Novelty: Old | ConceptID: Met3

20
Several researchers have demonstrated heterogeneous microfluidic immunoassays based on immobilized antibodies.3,6,12

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met4

21
Microfluidic bioanalytical assays that operate without an immobilized phase have also been demonstrated.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met5

22
Such approaches include an assay based on the rate of diffusion of antibody-antigen complexes in solution17,18 and a technique for maintaining beads in place in a recirculating flow stream without permanently immobilizing them.19

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met5

23
The work presented here demonstrates the application of a reversible bead immobilization technique which we have recently described20 for the immobilization of immunoassay reagents within a microfluidic channel in an on-demand, spatially and temporally controlled, and reversible fashion.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con4

24
The digoxin antibody–antigen couple was used as a model competitive immunoassay system21-24 to show how temperature-responsive bioanalytical beads can be utilized to couple the capture and release of target analytes for quantitative analysis.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met6

Experimental

Materials

25
Digoxin and the biotinylated monoclonal murine anti-digoxin IgG (clone DI-22) were obtained from Sigma (St. Louis, MO).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

26
BODIPY® FL digoxigenin was obtained from Molecular Probes (Eugene, OR).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

27
Wild-type streptavidin was expressed and purified according to a previously published protocol.25

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

28
The N-hydroxysuccinimidyl ester of poly(N-isopropylacrylamide) (NHS-PNIPAAm) was synthesized according to a previously published protocol.26

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

29
100 nm diameter Polybead® Amino Microspheres from Polysciences (Warrington, PA) were co-modified with PNIPAAm and 3.4 kDa poly(ethylene glycol)–biotin (PEG-b) (Shearwater Corporation, Huntsville, AL) as previously described.20

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

30
PET sheets used in the fabrication of the microfluidic device were obtained from Fralock, Inc. (San Carlos, CA).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

Preparation of antibody-coated beads

31
The antibody-bearing beads used in this experiment are described schematically in Fig. 1a.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

32
PNIPAAm and PEG-b doubly modified beads were incubated 15 minutes with equimolar (relative to immobilized biotin groups) quantities of streptavidin in pH 7.6 phosphate buffered saline (PBS, 50 mM phosphate, 5 mM NaCl).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp2

33
To remove any unbound streptavidin, the beads were centrifuged at 16 600g for 15 minutes at 4 °C.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp2

34
The supernatant was removed and the beads were resuspended in pH 7.6 PBS.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp2

35
This suspension was vortexed and agitated at 4 °C for 2 hours to assure that no bead aggregates remained.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp2

36
Biotinylated anti-digoxin IgG was then loaded onto the beads by adding it to the streptavidin-coated bead suspension at a 20-fold molar excess of streptavidin (assuming streptavidin tetramers bound at a 1 : 1 stoichiometry to biotin moieties in the first step).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp2

37
The bead suspension injected into the microfluidic channel consisted of 0.10 wt% doubly modified b-PEG/PNIPAAm beads loaded with streptavidin and anti-digoxin IgG, 0.40 wt% singly modified PNIPAAm beads, and 2.5 mg mL−1 free PNIPAAm in pH 7.6 PBS.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

38
The singly modified PNIPAAm beads and free PNIPAAm were added to decrease the concentration of doubly modified beads required to form a continuous adherent network on the channel walls, and to thereby conserve protein reagents.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

39
The suspension was degassed by agitation under vacuum for approximately five minutes immediately prior to use.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

Microfluidic device

40
Experiments took place in what was described in our previous publication as a “type A” channel.20

Type: Method | Advantage: None | Novelty: New | ConceptID: Met7

41
Briefly, the device was constructed from stacked layers of laser-machined PET.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp4

42
It consisted of a primary channel with one input port and one output port, with dimensions of 54 mm × 4 mm (at the widest point in the channel) × 300 µm.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp4

43
A small injection channel for introducing smart bead suspension led into the primary channel.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp4

44
Temperature control was accomplished via incorporation of a thin-film Therma-Clear™ heater and a HeaterStat™ electronic feedback controller (both from Minco, Minneapolis, MN).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp4

45
When the heater was activated, the controller maintained the temperature in a 20 mm-long region of the channel between 33 and 37 °C (this range of temperatures reflects the precision of the controller).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp4

46
In experiments, the heater was activated to trigger the bead aggregation caused by hydrophilic-to-hydrophobic PNIPAAm phase transition and later deactivated to reverse that process.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp4

47
The assembled device was mounted in a microfluidic manifold incorporating an aluminium pressure plate and polydimethylsiloxane (PDMS) gaskets to seal the input and output ports of the device to polyether ether ketone (PEEK) tubing.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp4

48
Computer-controlled syringe pumps (Kloehn Co., Ltd., Las Vegas, NV) were used to drive the flow through the devices, as described in our previous publication20.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp4

Digoxin immunoassay

49
The immunoassay protocol is described schematically in Fig. 1b.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod2

50
Two hundred microlitres of the mixed bead suspension were injected via the bead injection channel, connected by the manifold to a short length of PEEK tubing.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

51
After bead injection, this tubing was closed off with a standard HPLC tubing valve, preventing flow through the injection channel.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

52
The input port of the primary channel was connected to the output of a flow injection valve that allowed the operator to select between two flow sources: a 0.3 mL sample loop and a 1.5 mL sample loop.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

53
The 1.5 mL sample loop was filled with degassed PBS.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

54
The 0.3 mL sample loop contained BODIPY® FL digoxigenin in degassed PBS at a concentration of 2.5 µM mixed with digoxin at a concentration of 0, 1.28, 2.56, or 12.8 µM.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

55
The heater was activated in the absence of flow for 10 minutes.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

56
The pumps were then activated, pushing buffer from the 1.5 mL sample loop through the system at a rate of 20 µL min−1.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

57
Fluid flowing through the device was captured at the exit of the manifold output tubing in 50 µL aliquots.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

58
After 2.5 minutes of buffer wash, the injection valve was switched to the 0.3 mL sample loop, allowing the labeled digoxigenin/digoxin sample to flow into the device.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

59
The valve was switched back to the buffer-containing sample loop after 1.25 minutes, making the volume of the digoxigenin/digoxin sample 25 µL.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

60
After allowing the sample to bind and flow-through to be washed out (27.5 minutes after the initiation of flow), the heater was deactivated while the flow was maintained at a constant rate.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

61
Aliquots were collected for an additional 15 minutes as the aggregated beads dissolved.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

62
The output aliquots were then diluted to 500 µL in deionized water and the fluorescence of each sample was measured on a Perkin Elmer LS50B fluorescence spectrophotometer (Perkin Elmer Instruments, Inc., Shelton, CT), with excitation of the BODIPY fluorophore at 492 nm and emission at 511 nm.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

63
Experiments were performed in triplicate for each concentration investigated.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

64
The presence of beads in the output samples introduced a scattering signal in the fluorescence measurements.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp6

65
This signal made the measured fluorescence higher than it would be in a sample containing no beads.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp6

66
To correct for this effect, a set of samples containing a range of bead concentrations (determined by the OD600 of the samples) but no fluorescent species were analyzed in the fluorescence spectrophotometer.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp6

67
This analysis yielded a linear relationship between bead concentration and scattered fluorescent signal.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp6

68
For each immunoassay sample, the OD600 was measured and this linear relationship was used to determine what scattering signal to subtract from the measured fluorescence signal.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp6

Results and discussion

69
The bioanalytical system described in this study utilizes the temperature-responsive polymer poly(N-isopropylacrylamide) (PNIPAAm) to control the aggregation and “stickiness” of biofunctionalized beads.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met8

70
PNIPAAm is a “smart” polymer that undergoes a hydrophilic–hydrophobic phase transition at temperatures above the lower critical solution temperature of approximately 28 °C in aqueous buffer.27

Type: Method | Advantage: None | Novelty: New | ConceptID: Met8

71
The phase transition is accompanied by aggregation and precipitation of PNIPAAm molecules.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met8

72
Several authors have reviewed the application of this property to the fields of biotechnology and biomedicine.28-31

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac1

73
We have previously shown that when PNIPAAm-coated latex beads are also modified with a biotin–PEG moiety, they can be used to form a temperature-sensitive microfluidic chromatographic resin capable of separating the biotin-binding protein streptavidin from a flow stream.20

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac1

74
This system has been further developed here to demonstrate its capacity to provide the key elements of an in-channel immunoassay.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj2

75
The injectable immunoassay system is described schematically in Fig. 1.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj2

76
Fig. 1a is a diagram of the antibody-bearing beads.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

77
The 100 nm amine-coated polystyrene latex beads were modified using NHS-ester chemistry with two separate species: 11 kDa PNIPAAm and 3.4 kDa biotin-PEG.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met9

78
Approximately 40% of the amine groups on each bead (ca. 20 000 per bead) were biotinylated, as measured by the 2-(4′-hydroxyazobenzene) benzoic acid (HABA) assay.32

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs1

79
Streptavidin was then complexed to the beads, followed by complexation of the biotinylated anti-digoxin IgG to the free streptavidin binding sites.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met10

80
The digoxin/digoxigenin antigen system was used as a model for demonstrating the key steps of a competitive immunoassay.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met11

81
A schematic of the model protocol is provided in Fig. 1b.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod2

82
The stimuli-responsive beads displaying the anti-digoxin antibody (step 1) were heated above the LCST of the grafted PNIPAAm to immobilize the aggregated bead network on the channel surface in the heated region (step 2).

Type: Method | Advantage: None | Novelty: New | ConceptID: Met12

83
Channel temperature control was accomplished using a system that maintained the channel temperature between 33 and 37 °C when activated, as determined by placing a thermocouple in the channel under no-flow conditions.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met12

84
Though the temperature cannot be expected to be uniform throughout the channel, PNIPAAm phase transition occurs over a very narrow temperature range, and the polymer can be expected to be in its hydrophobic phase so long as the local temperature is above ∼28 °C under these buffer conditions.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met12

85
Following heating and wash through of unbound beads (step 3), a sample containing both the target antigen molecule (digoxin) and a fluorescently labeled antigen analog (BODIPY® FL digoxigenin) was flowed through the channel (steps 4 and 5).

Type: Method | Advantage: None | Novelty: New | ConceptID: Met12

86
A constant concentration of the labeled digoxigenin was used in all experiments.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met12

87
Since the two species in the sample compete for antibody binding, the amount of labeled digoxigenin that binds depends on the concentration of target digoxin in the sample.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met12

88
The concentration of digoxin in a given sample can therefore be determined by tracking the amount of antibody-bound fluorescent digoxigenin.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met12

89
After the sample had been allowed to flow through the channel, the heater was deactivated, lowering the channel temperature below the PNIPAAm LCST and causing the beads to disaggregate and leave with the flow stream (step 6).

Type: Method | Advantage: None | Novelty: New | ConceptID: Met12

90
Any antibody-bound digoxin or digoxigenin molecules left the channel with these beads.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met12

91
Throughout this procedure, 50 µL aliquots were collected at the channel output for subsequent fluorescence analysis.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met12

92
The successful competitive capture of digoxin and digoxigenin is shown in Fig. 2.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res3

93
The fluorescent intensities of the system output fractions are given as fractions of total fluorescence measured over the course of the experiment.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met13

94
The zero point on the x-axis corresponds to the initiation of pump-driven flow, after bead aggregation and adhesion had been allowed to take place in the heated channel.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met13

95
The initially elevated fluorescence values at the 50 and 100 µL points are due to non-adherent beads being washed out of the channel.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res4

96
Previous work has shown that approximately 50% of the thermally immobilized bead suspension adheres to the channel walls under flow conditions.20

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac2

97
Given the ratio of antibody loading on the beads, this implies that approximately 600 picomoles of antibody are available on the channel surfaces for binding to digoxin/digoxigenin.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res5

98
This is approximately a ten-fold excess relative to the total amount of labeled digoxigenin injected.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res5

99
The peaks that begin at 250 µL represent unbound BODIPY® FL digoxigenin flowing past the immobilized antibodies and out of the channel.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res6

100
At the 550 µL point, the heater was deactivated, lowering the temperature in the channel and resulting in the dissolution of the adherent bead matrix.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res7

101
The beads were washed out of the channel, along with the bound fractions of digoxin and BODIPY® FL digoxigenin.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res7

102
Previous work has shown the process of bead release upon decrease in channel temperature to be near 100% efficient.20

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac3

103
The fluorescence signal from the bound digoxigenin is observed as the peak beginning at the 600 µL point.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res8

104
As digoxin concentration increases, the flowthrough peak at 250 µL becomes larger while the elution peak at 600 µL becomes correspondingly smaller.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs2

105
This change is due to digoxin competition with fluorescent digoxigenin for binding to the immobilized antibodies.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res9

106
As less labeled digoxigenin binds the beads, more passes through the channel with the flowthrough peak (labeled A on Fig. 2), and less flows out with the beads in the elution peak (B).

Type: Result | Advantage: None | Novelty: None | ConceptID: Res9

107
The errors at each individual point in Fig. 2 make it difficult to distinguish between digoxin concentrations.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res10

108
However, by considering total peak area, varying digoxin concentrations can be easily discriminated, as shown in Fig. 3.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res11

109
The plot in Fig. 3a corresponds to the flowthrough peak, while Fig. 3b corresponds to the elution peak.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs3

110
Peak areas were obtained by simply integrating the peaks shown in Fig. 2, after drawing straight lines between the data points.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met14

111
The flowthrough peak was integrated from the 200 µL point to the 450 µL point, while the elution peak was integrated from the 550 µL point to the 700 µL point.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met14

112
While the 1.28 and 2.56 µM points remain statistically indistinguishable in both plots, the 0 and 12.8 µM points are distinguishable from all other points.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res12

113
In this model device format, concentrations of digoxin as low as 1.28 µM could be reliably determined and varying concentrations of digoxin ranging over an order of magnitude could be distinguished.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res13

114
The flowthrough peak data were sufficient to provide the full calibration curve throughout this digoxin concentration range.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res13

115
These results demonstrate that this system is capable of reporting the competitive binding and measurement of a target antigen in a format that can conveniently provide a wide range of concentration dependences.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con5

116
Because the fluorescence measurements were made on arbitrary flow-through aliquots (related to the channel dimensions and volumes) that were subsequently diluted to accommodate measurements in a standard benchtop fluorometer, the particular accessible range of digoxin concentrations reported in this study does not represent the intrinsic sensitivity of this system.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con6

117
That sensitivity will ultimately be a function of the device and detection design, and on-chip detection schemes and optical probes that minimize dilution and maximize signal should provide particularly important sensitivity enhancements.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con6

118
Finally, it is important to note that since the process of bead adhesion to the channel wall is not stoichiometric, the amount of immobilized antibody is expected to vary slightly with each trial.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con7

119
This imprecision results in the relatively large error bars seen in Fig. 2.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con7

120
However, the data presented in Fig. 3 demonstrate that meaningful results can be extracted from this platform even in light of the variations in amount of antibody immobilized.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con7

Conclusions

121
The reversible biomolecular immobilization facilitated by this technique allowed for all experiments to be performed in a single device over a period of several weeks with a fresh immobilized antibody substrate for each new experiment.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res14

122
Loading this substrate took only ten minutes, and it was not necessary to remove the device from the fluidic manifold connecting it to the sample delivery system.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met15

123
This platform is completely general in that any molecule that can be biotinylated or otherwise bound to the surface of a latex bead (e.g. enzymes, ligands, oligonucleotides) can be immobilized in microfluidic channels via this technology.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con8

124
Finally, the precise and reversible temperature-responsive nature of the smart polymer gives engineers a novel means of control for microfluidic processes, since the spatial distribution of immobilized molecules in a microfluidic device can be controlled merely by controlling the temperature at various locations in the device.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con9