Fluorescence Detector for Capillary Separations ... - ACS Publications

Nov 26, 2014 - Fluorescence Detector for Capillary Separations Fabricated by 3D ..... Open-Source-Based 3D Printing of Thin Silica Gel Layers in Plana...
1 downloads 0 Views 2MB Size
Download PDF
Technical Note pubs.acs.org/ac

Fluorescence Detector for Capillary Separations Fabricated by 3D Printing Jan Prikryl and Frantisek Foret* Institute of Analytical Chemistry AS CR, v. v. i., 60200 Brno, Czech Republic S Supporting Information *

ABSTRACT: A simple inexpensive light-emitting diode (LED)-based fluorescence detector for detection in capillary separations is described. The modular design includes a separate high power LED source, detector head, designed in the epifluorescence arrangement, and capillary detection cells. The detector head and detection cells were printed using a 3D printer and assembled with commercially available optical components. Optical fibers were used for connecting the detector head to the LED excitation source and the photodetector module. Microscope objective or high numerical aperture optical fiber were used for collection of the fluorescence emission from the fused silica separation capillary. As an example, mixture of oligosaccharides labeled by 8-aminopyrene-1,3,6-trisulfonate (APTS) was separated by capillary zone electrophoresis and detected by the described detector. The performance of the detector was compared with both a semiconductor photodiode and photomultiplier as light sensing elements. The main advantages of the 3D printed parts, compared to the more expensive alternatives from the optic component suppliers, include not only cost reduction, but also easy customization of the spatial arrangement, modularity, miniaturization, and sharing of information between laboratories for easy replication or further modifications of the detector. All information and files necessary for printing the presented detector are enclosed in the Supporting Information.

A

A variety of different optical arrangements of a sensitive fluorescence detector for microcolumn systems have been described as recently reviewed by Johnson and Landers,15 by Gonzalez and Laserna,16 or by Xiao et al.17,18 Besides, the efficient sample excitation and collection of the emitted fluorescence minimization of the system background is of critical importance for achievement of high detection sensitivity. Spurious background is typically a result of scattered excitation radiation, fluorescence of impurities, and Raman bands of the solvent. Background reduction strategies differ according to the background origin and involve spatial (iris diaphragm, pinhole) or optical (based on wavelength, polarization, or photobleaching) filtering or based on discrimination of fluorescence lifetimes of analytes vs background impurities. In the orthogonal arrangement with paths of excitation and emission beams at a 90° angle, separate lenses (microscope objectives) are employed for focusing of the excitation beam into the capillary and for collecting of the fluorescence.19 The main background contribution consists of the scatter of the laser light on capillary walls and commonly is suppressed spatially by the iris diaphragm and optically by a set of filters. In addition, changing of the 90° angle to the Brewster angle can be used to minimize the scatter.20−23 This detector architecture is

dditive manufacturing, also known as 3D printing, rapid prototyping, or solid-freeform technology, has become an efficient method for rapid prototyping and fabrication applications ranging from small home projects to architecture and industrial machining.1,2 The additive manufacturing sometimes marked as the next industrial revolution3,4 has already demonstrated capabilities as diverse as making prosthetic arms for children of war,5 fabrication of the hybrid exoskeleton robotic suit,6 custom-made implants and tissue scaffolds,7−9 or fabrication of fluidic devices for applications in synthetic10 and analytical chemistry.11 The history and basic principles of additive manufacturing, such as stereolitography, inkjet printing, selective laser sintering, laminated object manufacturing or fused deposition modeling (FDM), and its interesting applications in biotechnology and chemical sciences have recently been reviewed by Gross et al.12 While standard machining technologies will continue to dominate in areas requiring a wider range of materials and top fabrication precision, additive manufacturing allows fabrication from various materials depending on the used method with resolution down to 1 μm.12 The inexpensive FDM provides production of functional parts from available plastic materials with the required resolution and accuracy in the 10− 100 μm range.13,14 Fluorescence, especially laser or LED induced, represents one of the most frequently used selective and sensitive detection modes for capillary separations with sensitivity in the femtomole range or lower. © XXXX American Chemical Society

Received: June 25, 2014 Accepted: November 10, 2014

A

dx.doi.org/10.1021/ac503678n | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Technical Note

Figure 1. 3D printed fluorescence detection head scheme: the horn shaped hole H minimizes the excitation beam backreflection (A). Detail of the capillary guide: sample cuvette (B). The printed and assembled detection system including the capillary holder with mounted fused silica capillary (C). The LED source in an aluminum housing (D).

for a modular system with a separate excitation source (LED module), universal detection head, and capillary holder (detection cell). Individual modules, including the excitation source, detector head, and photodetector electronics, were connected using a commercial SMA connector terminated, optical fibers, and fiber-optic collimators. The detection head was designed in a standard epifluorescence arrangement for use with commercial (1/2 in. diameter) filters and allowed using attachment of either a microscope objective or fiberoptic collimator on the detection cell side. The detection head, cover lid, and capillary holder (detection cells) were designed in the user-friendly 3D-modeling software SketchUp (Trimble Navigation, Ltd.) and fabricated using the FDM printer EASY3DMAKER (AROJA, s. r. o., Czech Republic) from black polylactic acid (PLA). Source files (SketchUp and STL files) used for designing and printing of components are enclosed as Supporting Information. Two main advantages of the 3D printing are the simplicity and speed of the design and the possibility to create complex shapes inside closed objects, which would be difficult or impossible to fabricate by standard machining. Depending on the complexity, the design drawing (needed for any machining method) takes minutes to hours. The presented components took about 3 h to draw. The printing time depends on the type of the printer. We have used an inexpensive printer (∼2000 U.S. dollars (USD)) and the most complex part, the detector head, was printed in about 4 h. This compares well with standard machining, which would take longer. An example of complex internal shape, which would be difficult or impossible to create by standard machining using a mill, lathe, or drill is the horn shaped hole H minimizing the excitation beam backreflection, Figure 1. Originally, we have machined one detector head from an aluminum block and had it black anodized only to realize that modifications were needed during the measurements. The material (PLA or ABS) is inexpensive at ∼30 USD per kg. This allows late modifications without much additional expenses. In summary, the cost of the 3D printed detector head and detection cell was about 5 USD.

quite space-consuming because of separate excitation and emission parts. Confocal arrangement, profiting from usage of the same objective lens for sample excitation as well as for collection of the fluorescence, is able to restrict the emission region by using a micrometer-sized pinhole; therefore, only fluorescence of sample without laser scatter is collected.24,25 Minimizing the detection volume is also beneficial since it suppresses the effect of Raman scatter of the solvent. This is especially important for achieving the single molecule sensitivity; however, a system with precise pinhole optics is typically more expensive. An alternative way for elimination of the scattered light and spurious fluorescence from the capillary material is use of the sheath-flow cuvette approach.24,26−30 A variety of alternative approaches for construction of low-cost fluorescence detectors can be found in the literature.31−39 The epifluorescence design allows construction of compact and sensitive detectors suitable for miniaturized systems including microfluidic devices or capillary techniques.40 Here we are describing fabrication of a low-cost LEDinduced fluorescence (LED-IF) detector head using a 3D printer allowing fast, yet relatively precise (80 μm resolution) fabrication of complex shapes. Compared to the more expensive alternatives using the standard optic component, the 3D printing provides significant cost reduction and, especially, sharing of information between laboratories for easy replication or further modifications, customization of the spatial arrangement, and miniaturization.

■

EXPERIMENTAL SECTION 3D Printing of Parts. The main design criteria relate to the intended use for detection in fused silica capillaries. While we wanted to minimize most of the regular machining we also wanted a mechanically robust and stable setup with easy alignment and options for connecting different excitation sources, capillary detection cells, and detectors. On the basis of the previous experience with fiber-optics,41,42 we have opted B

dx.doi.org/10.1021/ac503678n | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Technical Note

can be replaced without readjustment. It should be stressed that the printer resolution does not influence the alignment. The adjustment was as follows. The fused silica capillary was filled with water, and its accurate position relative to the microscope objective was set by changing the position of the capillary holder. During the adjustment, the image created by the excitation beam was observed on a paper screen. The observed pattern should be identical with or without the capillary inserted into the holder. Under these conditions, the capillary is placed accurately in the focal point of the microscope objective. Once adjusted, the capillary holder position was fixed to the detection head by screws as shown in Figure 1. Determination of the Limits of Detection (LODs). Testing concentration series were prepared by diluting stock solution of fluorescein (Reactifs RAL, Martillac, France) and sodium tetraborate decahydrate (LACHEMA, Brno, Czech Republic) (10 mM, pH = 9.3) in water-methanol solution (25% (v/v) of 99.8%, PENTA, Chrudim, Czech Republic). The signal from each fluorescein concentration was measured after flushing the capillary with pure methanol. The signals were recorded as an average of three measurements in bare fused silica capillary at a flow rate 8 μL min−1 starting from the lowest fluorescein concentration. Signal was acquired by the DAQ system U-PAD2 (DataApex, Praha, Czech Republic) at sampling frequency of 12.5 Hz. Capillary Electrophoresis. Simple capillary holder for CZE was also fabricated by 3D printing (Figure S-1 in the Supporting Information, skp and stl file are enclosed). CZE was performed in a polyacrylamide-coated fused silica capillary (effective length 49.5 cm, total length 59.5 cm) filled with 100 mM acetic acid (pH = 2.8) as background electrolyte. Preparation of the dextran ladder by partial hydrolysis of dextran was described elsewhere.43 Samples (diluted APTSlabeled dextran ladder, respectively, solutions of APTS standard) were injected hydrodynamically by hydrostatic pressure (980 Pa) for 20 s corresponding to an injected volume of 26 nL or 1.2% of the effective length of the capillary. High-voltage power supply CZE1000R (Spellman High Voltage Electronics Corp., Plainview, NY) was used in constant voltage mode at 14 kV.

On the basis of the prices of the local machine shop we have saved about 500 USD. The cost of commercial mechanical parts as described in ref 15 is 580 USD. Since several such detectors are being used in our lab, the initial cost of the 3D printer has already paid off. In addition, modifications for different applications/space arrangements are easy as shown here by printing two different detection cells with either a microscope objective or optical fiber for excitation/fluorescence collection. Optical Components. Fused silica capillary of inner diameter of 75 μm (Polymicro Technologies, Phoenix, AZ) was used as a sample cuvette or separation capillary. Optical elements, including a microscope objective (40×, 0.65 N.A.) and optical filters (no. 62-976 long-pass filter, o.d. 4, 500 nm, diameter 12.5 mm; no. 69-866 dichroic long-pass filter, 500 nm, diameter 12.5 mm; no. 84-693 short-pass filter, o.d. 4, 500 nm, diameter 12.5 mm) were purchased from Edmund Optics GmbH, Karlsruhe, Germany. Blue-emitting LED with the emission maximum at 470 nm (spectral bandwidth at 50%, Δλ = 22 nm) (OD-469L, Opto Diode Corp., Newbury Park, CA) was used as an excitation source. The LED was housed in an aluminum cooling profile with thermal resistance of 7.5 K/W (CHL34C/50, EZK, Roznov pod Radhostem, Czech Republic). The fluorescence emission was detected either by a photodiode (ODA-6WB-500M, Opto Diode Corp.) or by a photomultiplier tube (R647, Hamamatsu Photonics K.K., Hamamatsu, Japan). The photodiode included a built-in transimpedance amplifier with 500 MΩ precision internal resistor and the signal was further amplified 30× prior to recording the signal resulting in the total conversion of 10 V nA−1. The excitation and emission radiation was guided by optical fibers (M40L01, diameter 400 μm, 0.48 N.A., Thorlabs GmbH, Dachau/Munich, Germany) coupled via fiber optic collimators (F230SMA-A 543 nm, f = 4.34 mm, NA = 0.57, Thorlabs). All optical components are specified in detail in Table S-3 in the Supporting Information. Complete electrical scheme of the detector is also in the Supporting Information (Figure S-3). Detector Assembly. The detector head printed on the 3D printer consisted of the main body, containing slots for optical filters and holes for optical fiber collimators, microscope objective, and the cover plate assuring light tight assembly. Since the resolution as well as minor dimension deformations common to all thermal 3D printers do not allow for reliable printing of precise threads standard screw-taps (specified in Table S-3 in the Supporting Information) were used to set up the microscope objective and optical fiber collimators in the detector head. Optical filters were inserted into slots without any further machining and secured in position by the detector head lid. The holder of the fused silica capillary was also 3D printed and attached to the detector head by adjustable screws. This is important for precise positioning of the capillary relative to the microscope objective for achieving best results. Instead of the commonly used translation stages we have used stainless steel capillary guides permanently glued to the capillary holder. The 1 cm long capillary guides, prepared from a 22 gauge (internal diameter 413 μm) syringe needle, provided firm positioning of the capillary within the spot size of the excitation beam (Figure 1B). The spot size illuminating the capillary was determined by the optical fiber diameter (400 μm). A smaller spot size can be achieved either using a smaller diameter optical fiber or additional optics; however, in both cases the total excitation power at the detection window would be reduced. Once the holder is aligned by the adjustable screws the capillary

■

RESULTS AND DISCUSSION Hardware Setup. The detector scheme is in Figure 1A. The detector head, the lid, and the capillary holder were printed from black PLA (Figure 1B). PLA, a biodegradable thermoplastic, was chosen after preliminary tests for its low (unmeasurable) background fluorescence. In addition, the material has very high absorbance even in very thin layers, assuring perfect light tightness of the whole assembly. The LED was operated in the constant current mode with 270 mA supplied by a constant current source with the LM 317 regulator connected as precision current limiter according to the manufacturer (Texas Instruments, Dallas, TX). The measured output power of the LED was 65 mW. Excitation radiation provided by the blue LED was coupled by input collimator into the optical fiber and launched into the collimator of the detector body as a nearly parallel beam. While the LED could be incorporated directly into the detector head, we have opted for separating it into an independent unit. Such an arrangement minimizes the thermal fluctuations of the detector head due to the heating of the LED and makes the setup more flexible in space limited projects. The total power coupled from the LED into the 400 μm optical fiber was C

dx.doi.org/10.1021/ac503678n | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Technical Note

the corresponding LOD is about 70 pM which is on the same order of magnitude as our detection system.52 Comparable LODs were achieved by de Jong and Lucy who presented a LOD of 3 pM for fluorescein in flushing mode and 25 pM for fluorescently labeled BSA in separation mode using a high-power LED (500 mW) and a more complex and spaceconsuming orthogonal instrument.53 While the sensitivity of the presented simple system could be further improved by employing higher quality filters and increasing the excitation power, it would also lead to increased cost of the detector. Demonstration of the Detector for CZE Analysis. The setup with the photodiode detector was used for CZE−LED-IF analysis of APTS-labeled dextran ladder (Figure 2). Products of

measured to be 2.6 mW at the capillary window. As noted in the Detector Assembly section, the position of the detection capillary was fixed and did not need any adjustment when changed. Inside the detector head, the light passed through a short pass filter for suppression of LED emission above 500 nm before reaching the dichroic mirror reflecting light below 500 nm into the microscope objective. The part of the excitation beam passing the dichroic mirror was captured by multiple reflections at the black PLA surface inside the horn shaped hole H (beam trap) minimizing its backreflection toward the detector. The entrance diameter of the beam trap was selected about 500 μm wider than that of the excitation beam. The curvature was selected empirically, to reflect a parallel excitation beam toward the narrowing end of the beam trap. The objective focused the excitation radiation into the fused silica capillary held in place by an adjustable capillary holder. Fluorescence was collected by the same microscope objective and passed through the dichroic mirror to the long pass filter blocking the scattered excitation radiation. Next, after passing through the long pass filter the fluorescence was collimated into the output optical fiber and detected by a photodiode or a PMT. Photographs of the assembled detector are in Figure 1C. LODs Determination. The performance of the assembled detector and LODs were evaluated either with the photodiode or PMT detectors. In addition, the excitation/fluorescence beams were coupled to the capillary either by using the microscope objective (40×, 0.6 N.A.) or a high numerical aperture (0.48 N.A.) optical fiber. During the measurements, the capillary was continually rinsed with fluorescein solutions (concentrations from 5 × 10−10 M to 5 × 10−8 M) at the flow rate of 8 μL s−1. Continuous sample flow was selected to prevent effects of photobleaching. LODs were calculated as a 3fold standard deviation of six blank solutions signals, and concentrations were calculated from calibration curves. During the experiments, the LODs were determined to be 92 pM with detection by the photodiode and 10 pM with the photomultiplier tube. While the PMT sensitivity is superior to the one with the photodiode detector, the photomultiplier setup is also significantly more expensive. The sensitivity of the photodiode setup is limited, mainly it is electronic noise and, in principle, could be further improved only by increasing the excitation power. On the other hand, the PMT setup is limited mainly by the residual excitation radiation and it’s sensitivity could very likely be improved by using better optical filters. This would also significantly add to the cost of the system beating the original purpose of the inexpensive system. Regarding comparison to recently published setups, the most sensitive fluorescence systems described in the literature are based on the sheath-flow cuvette with laser excitation, where the LOD for FITC-labeled (fluoresceinisothiocyanate-labeled) arginine reached 5 pM,26 for 100-mer fluorescein-labeled oligonucleotide was 20 pM,44 for tetramethylrhodamine isothiocyanate a detected amount of 300 molecules,27 and for sulphorhodamine 101, 6.1 ± 1.3 molecules were detected.29 Setups based on using LEDs as an excitation source achieved concentration LODs typically in the nanomolar range37,45−51 and were reviewed in refs 17 and 18. The most similar epifocal detection system designed for coupling nano-LC to a microfluidic device with bioaffinity analysis was built by Heus et al. Sensitivity of this LED-based epifluorescence system with a quartz microscope objective (20×), and PMT was determined for 500 pM solution of rhodamine 110 as the signal-to-noise ratio which was 23. Assuming linearity of the calibration curve,

Figure 2. Electropherogram of a dextran ladder sample labeled by APTS. CE conditions are described in detail in the Experimental Section.

dextran hydrolysis labeled by APTS were separated, and the LOD for APTS label was determined in CZE mode. As noise, the standard deviation of 80 s baseline sections of six APTS samples was taken. The LOD calculated from the calibration curve based on the height of APTS peaks was 800 pM. In comparison to fluorescein, the increased LOD was due to different fluorescence spectra (fluorescein better fits the LED spectrum and the emission filter, Figure S-2 in the Supporting Informatiuon).54−56 Further contribution is the lower APTS quantum yield compared to fluorescein.57,58 It is worth noting that LODs in capillary electrophoresis are strongly influenced by the injection procedure (see Figure S-3 in the Supporting Information); therefore, LODs for fluorescein and APTS are not directly comparable. While fluorescein provides better detection sensitivity with the presented detector (see the previous section) we have selected the APTS labeled oligosaccharides for demonstration of both the detector sensitivity (sufficient for noncritical analyses) and its performance in high-efficiency separations. Separation efficiency of oligosaccharides zones (∼150 000 plates for peak 10) was comparable to the resolution obtained using the laboratorybuilt CZE−LIF system with a 488 nm Ar-ion laser and a PMT used in our laboratory. Optical Fiber in Place of the Microscope Objective. In some cases, spatial limitations require miniaturization of the detection cell. A typical example includes incorporation of the D

dx.doi.org/10.1021/ac503678n | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Technical Note

fluorescence detector into commercial capillary cartridges. In such a case, optical fibers can be used for bringing the excitation radiation into the capillary cartridge and collecting the emission. Often a bifurcated fiber optic bundle is used to improve the collection efficiency.59,60 Here we have tested the detector setup where the microscope objective was substituted by a high numerical aperture SMA terminated optical fiber serving as a guide for excitation light as well as for the fluorescence. The detection cell consisted of two rectangular black PLA plates with capillary positioned in between and the optical fiber directly touching the capillary with removed polyimide coating. In this setup the SMA adapter for the SMAterminated optical fiber was attached to one of the PLA plates and two stainless-steel capillaries (1 cm long capillary guides, prepared from a 22 gauge syringe needle), serving as the separation capillary guide, were glued on this PLA plate. Both plates were connected by screws spaced by a black rubber Oring (Figure S-2 in the Supporting Information). The assembled detection cell could fit into any commercial capillary cartridge and was robust without any need for additional setting. Fluorescein LODs for this arrangement were determined using the same procedure as described above for arrangement with a microscope objective and were 280 pM using PD and 230 pM using PMT, respectively. When compared to the arrangement with the microscope objective, spatial filtering by optical fiber was less effective than by the microscope objective. As a result, more scattered light reached the detector resulting in higher background signal. This, in turn, did not allow application of more than 400 V on the PMT resulting in the limited sensitivity. Thus, the LOD achieved by PD was practically the same as the LOD achieved with PMT (Table S-1 in the Supporting Information). It is worth mentioning that the stability (short-term noise and drift) of the LED is more than an order of magnitude better than that of the Ar ion laser. Thus, the residual background scatter is less detrimental than in the laser based system. Again, improvements could be made using better optical filters at the cost of increased price. It is worth mentioning that while less sensitive, this setup is very universal since it allows fluorescence measurements in very confined spaces.

considered instead of LED when the desired wavelength is available. Further improvements in sensitivity could be achieved with better (more expensive) optical filters and PMT or spectrograph based CCD detectors, providing additional spectral information. However, this might beat the original purpose of simplicity and cost. Complete description of the assembly and the files for the 3D printing are available in the Supporting Information.

■

ASSOCIATED CONTENT

S Supporting Information *

Additional tables and figures. The zip file contains .skp and .stl files that are for a 3D printer to replicate the work done here. For more information regarding these files, please contact Frantisek Foret at [email protected]. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +420 541 212 113. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the Grant Agency of the Czech Republic (Grant P206/12/G014) and Institutional support RVO:68081715. Additional funding was provided by the Academy of Sciences of the Czech Republic (Grant M200311201) and by the European Social Fund and the state budget of the Czech Republic (Grant CZ.1.07/2.3.00/ 20.0182). We thank Jan Partyka for providing APTS-labelled dextran ladder.

■

REFERENCES

(1) 3ders.org. Applications. http://www.3ders.org/applications.html (accessed April 29, 2014). (2) 3D Printing Industry Limited. 3DP Applications, http:// 3dprintingindustry.com/3dp-applications/ (accessed April 25, 2014). (3) Barnatt, C. 3D Printing: The Next Industrial Revolution; CreateSpace Independent Publishing Platform: An Amazon Company, 2013. (4) Council, A.; Petch, M. 3D Printing: Rise of the Third Industrial Revolution (Gyges 3D Presents); gyges3d.com, 2014; p 116. (5) Not Impossible Labs. Project Daniel, http://www. notimpossiblelabs.com/#!project-daniel/c1imu (accessed April 25, 2014). (6) Ekso Bionics. 3D Systems and Ekso Bionics Announce First “Bespoke” Exoskeleton, http://www.eksobionics.com/pressrelease (accessed April 24, 2014). (7) Hoque, M. E.; Chuan, Y. L.; Pashby, I. Biopolymers 2012, 97, 83− 93. (8) Giannatsis, J.; Dedoussis, V. Int. J. Adv. Manuf. Technol. 2009, 40, 116−127. (9) Azari, A.; Nikzad, S. Rapid Prototyping J. 2009, 15, 216−225. (10) Symes, M. D.; Kitson, P. J.; Yan, J.; Richmond, C. J.; Cooper, G. J. T.; Bowman, R. W.; Vilbrandt, T.; Cronin, L. Nat. Chem. 2012, 4, 349−354. (11) Anderson, K. B.; Lockwood, S. Y.; Martin, R. S.; Spence, D. M. Anal. Chem. 2013, 85, 5622−5626. (12) Gross, B. C.; Erkal, J. L.; Lockwood, S. Y.; Chen, C.; Spence, D. M. Anal. Chem. 2014, 86, 3240−3253. (13) Stanek, M.; Manas, D.; Manas, M.; Navratil, J.; Kyas, K.; Senkerik, V.; Skrobak, A. Int. J. Math. Comput. Simul. 2012, 6, 550− 557.

■

CONCLUSIONS In this study, we presented a new approach for fabrication of a low-cost fluorescence detection head for capillary separation methods using a FDM 3D printer. FDM provides an excellent way to fabricate all mechanical parts with sufficient precision (80 μm in this work) and allows rapid modifications to be made during the design improvement. We have prepared two arrangements either with microscope objective or with high numerical aperture optical fiber. Both arrangement were tested for sensitivity and detection in capillary electrophoresis. Both arrangements are universal and can be used for different purposes where sensitive fluorescence detection in a limited space is needed. One of the main limitations of the LED based detectors in general is the limited power density (power emitted per unit surface area of the LED chip). Unlike lasers, which can be focused to diffraction limited spot sizes, the excitation power density at the detection cell cannot exceed that at the surface of the omnidirectional LED chip. In principle, LED chips with the same power but smaller dimensions might bring better detection sensitivities in the future. Of course, inexpensive lasers should be always E

dx.doi.org/10.1021/ac503678n | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Technical Note

(14) Chuk, R. N.; Thomson, V. J. Rapid Prototyping J. 1998, 4, 185− 196. (15) Johnson, M. E.; Landers, J. P. Electrophoresis 2004, 25, 3513− 3527. (16) Gonzalez, E.; Laserna, J. J. Quim. Anal. 1997, 16, 3−15. (17) Xiao, D.; Zhao, S. L.; Yuan, H. Y.; Yang, X. P. Electrophoresis 2007, 28, 233−242. (18) Xiao, D.; Yan, L.; Yuan, H. Y.; Zhao, S. L.; Yang, X. P.; Choi, M. M. F. Electrophoresis 2009, 30, 189−202. (19) Yeung, E. S.; Wang, P. G.; Li, W. N.; Giese, R. W. J. Chromatogr. 1992, 608, 73−77. (20) Kuhr, W. G.; Yeung, E. S. Anal. Chem. 1988, 60, 2642−2646. (21) Kuhr, W. G.; Yeung, E. S. Anal. Chem. 1988, 60, 1832−1834. (22) Lee, T. T.; Yeung, E. S. J. Chromatogr. 1992, 595, 319−325. (23) Larson, A. P.; Ahlberg, H.; Folestad, S. Appl. Opt. 1993, 32, 794−805. (24) Wu, S. L.; Dovichi, N. J. J. Chromatogr. 1989, 480, 141−155. (25) Hernandez, L.; Escalona, J.; Joshi, N.; Guzman, N. J. Chromatogr. 1991, 559, 183−196. (26) Cheng, Y. F.; Dovichi, N. J. Science 1988, 242, 562−564. (27) Chen, D. Y.; Swerdlow, H. P.; Harke, H. R.; Zhang, J. Z.; Dovichi, N. J. J. Chromatogr. 1991, 559, 237−246. (28) Chen, D. Y.; Dovichi, N. J. J. Chromatogr., B: Biomed. Appl. 1994, 657, 265−269. (29) Chen, D. Y.; Adelhelm, K.; Cheng, X. L.; Dovichi, N. J. Analyst 1994, 119, 349−352. (30) Chen, D. Y.; Dovichi, N. J. Anal. Chem. 1996, 68, 690−696. (31) Shrinivasan, S.; Norris, P. M.; Landers, J. P.; Ferrance, J. P. Clin. Lab. Med. 2007, 27, 173−+. (32) Novak, L.; Neuzil, P.; Pipper, J.; Zhang, Y.; Lee, S. H. Lab Chip 2007, 7, 27−29. (33) Wu, J.; Liu, X.; Wang, L.; Dong, L.; Pu, Q. Analyst 2012, 137, 519−525. (34) Geng, X. H.; Wu, D. P.; Guan, Y. F. Talanta 2012, 88, 463−467. (35) Xu, J.; Chen, S. H.; Xiong, Y.; Yang, B. C.; Guan, Y. F. Talanta 2008, 75, 885−889. (36) Xu, J.; Xiong, Y.; Chen, S. H.; Guan, Y. F. Talanta 2008, 76, 369−372. (37) Yang, F.; Li, X. C.; Zhang, W.; Pan, J. B.; Chen, Z. G. Talanta 2011, 84, 1099−1106. (38) Yao, M. J.; Shah, G.; Fang, J. Micromachines 2012, 3, 462−479. (39) Geng, X. H.; Wu, D. P.; Wu, Q.; Guan, Y. F. Talanta 2012, 100, 27−31. (40) Kuswandi, B.; Nuriman; Huskens, J.; Verboom, W. Anal. Chim. Acta 2007, 601, 141−155. (41) Foret, F.; Zhou, H. H.; Gangl, E.; Karger, B. L. Electrophoresis 2000, 21, 1363−1371. (42) Ryvolova, M.; Preisler, J.; Foret, F.; Hauser, P. C.; Krasensky, P.; Paull, B.; Macka, M. Anal. Chem. 2010, 82, 129−135. (43) Partyka, J.; Foret, F. J. Chromatogr., A 2012, 1267, 116−120. (44) Zhang, J. Z.; Chen, D. Y.; Wu, S.; Harke, H. R.; Dovichi, N. J. Clin. Chem. 1991, 37, 1492−1496. (45) de Jong, E. P.; Lucy, C. A. Anal. Chim. Acta 2005, 546, 37−45. (46) Sluszny, C.; He, Y.; Yeung, E. S. Electrophoresis 2005, 26, 4197− 4203. (47) Kuo, J. S.; Kuyper, C. L.; Allen, P. B.; Fiorini, G. S.; Chiu, D. T. Electrophoresis 2004, 25, 3796−3804. (48) Wang, S. L.; Fan, X. F.; Xu, Z. R.; Fang, Z. L. Electrophoresis 2005, 26, 3602−3608. (49) Yang, B. C.; Tan, F.; Guan, Y. F. Talanta 2005, 65, 1303−1306. (50) Zhao, S. L.; Yuan, H. Y.; Xiao, D. Electrophoresis 2006, 27, 461− 467. (51) Yang, B. C.; Tian, H. Z.; Xu, J.; Guan, Y. F. Talanta 2006, 69, 996−1000. (52) Heus, F.; Giera, M.; de Kloe, G. E.; van Iperen, D.; Buijs, J.; Nahar, T. T.; Smit, A. B.; Lingeman, H.; de Esch, I. J. P.; Niessen, W. M. A.; Irth, H.; Kool, J. Anal. Bioanal. Chem. 2010, 398, 3023−3032. (53) de Jong, E. P.; Lucy, C. A. Analyst 2006, 131, 664−669.

(54) Sharrett, Z.; Gamsey, S.; Hirayama, L.; Vilozny, B.; Suri, J. T.; Wessling, R. A.; Singaram, B. Org. Biomol. Chem. 2009, 7, 1461−1470. (55) Martin, M. M.; Lindqvist, L. J. Lumin. 1975, 10, 381−390. (56) Evangelista, R. A.; Liu, M. S.; Chen, F. T. A. Anal. Chem. 1995, 67, 2239−2245. (57) Magde, D.; Wong, R.; Seybold, P. G. Photochem. Photobiol. 2002, 75, 327−334. (58) Hartman, M. C. T.; Dcona, M. M. Analyst 2012, 137, 4910− 4912. (59) Hurth, C.; Lenigk, R.; Zenhausern, F. Appl. Phys. B: Lasers Opt. 2008, 93, 693−699. (60) Arraez-Roman, D.; Fernandez-Sanchez, J. F.; CortaceroRamirez, S.; Segura-Carretero, A.; Fernandez-Gutierrez, A. Electrophoresis 2006, 27, 1776−1783.

F

dx.doi.org/10.1021/ac503678n | Anal. Chem. XXXX, XXX, XXX−XXX