Volume 52, Number 12, 1998 APPLIED SPECTROSCOPY 16190003-7028 / 98 / 5212-1619$2.00 / 0

q 1998 Society for Applied Spectroscopy

SPECTROSCOPICTECHNIQUES

Interfacing A High-Sensitivity, Near-Infrared Laser-Induced FluorescenceDetector with a CommercialCapillary Electrophoresis Instrument

MENNO J. BAARS and GABOR PA-TONAY*Department of Chemistry, University Plaza, Georgia

State University, Atlanta, Georgia 30303

Index Headings: Near-infrared; Laser-induced ¯ uorescence; Ava-lanche photodiode detector; Capillary electrophoresis.

INTRODUCTION

Laser-induced ¯ uorescence (LIF) is the most sensitivemethod of detection available for use with capillary elec-trophoresis (CE).1 The majority of LIF applications todate have employed sample derivatization schemes usingvisible ¯ uorescent dyes. Uses of near-infrared (NIR) dyesfor analytical applications have been reported. 2±8 The ad-vantage of detection in the NIR region (about 670±1000nm) results from the lack of background interference,since few naturally occurring molecules can undergoelectronic transitions in this low-energy region of theelectromagnetic spectrum. Scatter (Rayleigh and Raman)can be a large contributor of noise at low analyte con-centrations and is reduced at higher wavelengths due toits dependence on the wavelength of detection by 1/ l 4.

Commercially available LIF detectors are optimizedfor the visible wavelength region, utilizing conventionallaser excitation sources and photomultiplier (PMT) de-tectors. Conventional laser sources are expensive andhave relatively short operating lifetimes. PMT detectorshave great sensitivity in the UV-visible region, but theirquantum ef® ciency rapidly decreases below 5% in thefar-red region. PMTs are quite expensive; they are easilydamaged by over exposure to light and have usable life-times around 10 000 hours. The large active surface areaof these detectors (cm 2) generally requires high magni-® cation optics when used with the 25 to 100 m m i.d.capillary detector windows.

Excitation in the NIR region can be accomplishedwith the use of compact and inexpensive GaAlAs laserdiodes. These laser diodes have stable and relatively high

Received 28 May 1998; accepted 5 August 1998.* Author to whom correspondence should be sent.

power outputs, and in contrast to other laser systems havelong operating lifetimes, low maintenance costs, and areeasy to use. 2,4,9±12 Detection can be achieved by usingsilicon avalanche photodiode (APD) detectors. These sol-id-state detectors offer quantum ef® ciencies around 80%in the NIR region, with low noise characteristics. Theyare rugged and have long usable lifetimes exceeding 100000 hours. APDs have a lower initial cost and low op-erating power requirements compared to PMTs. Thesmall active surface area of an APD ( , mm 2) is wellmatched to the capillary detector windows encounteredin CE, allowing incorporation of lower magni® cation op-tics in the instrumental design.

Several authors have reported on the use of ¯ uorescentNIR dyes for use with CE.13±15 The advantage of usingdiode lasers and avalanche photodiode CE detectors atwavelengths above 650 nm has been noted. 7,16±18 Thework completed to date involves the use of homemadeCE systems and detectors. This work presents a simpleinterface between a commercial APD-based near-infraredLIF detector and a commercial CE instrument. The sys-tem retains the fully automated injection, separation, anddata collection capabilities of the commercial CE instru-ment and is optimized for detection around 820 nm withlaser excitation at 787 nm.

EXPERIMENTAL

Reagents and Chemicals. IR-780 laser dye was pur-chased from Aldrich Chemical Co. NN382 dye was ob-tained as a gift from LI-COR Inc., Lincoln, NE. HPLC-grade methanol, ACS-grade borax, sodium phosphatemonobasic (monohydrate), and sodium phosphate dibasic(monohydrate) were purchased from Fisher Scienti® c,Fair Lawn, NJ. All water used was nanopure grade (Barn-stead Model D4751 ultrapure water system).

Instrumentation. The P/ACE 5000 capillary electro-phoresis (Beckman Instruments, Inc., Fullerton, CA) wasselected as the base of the NIR LIF system and was pur-chased from Beckman. An LIF cartridge with a 57 cmbare fused-silica capillary (50 cm to detector, 75 m m i.d.)was purchased with the instrument. The P/ACE 5000 hasbeen commercially available for many years and consistsof an automated capillary electrophoresis system withmultiple detector options. Both UV and LIF detectors areavailable, and detectors can be exchanged in about 30 sby using a simple and rugged mounting system. The re-movable detector mounting system lends the P/ACE 5000system to custom detector installation. Instrument oper-ation and data collection are fully automated through theBeckman ``System Gold’ ’ chromatographic software(version 8.01), run on a 486-33 personal computer.

A proprietary microscope and laser assembly manu-

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FIG. 1. Experimental instrument component diagram. The APD detec-tor, diode laser, and detector electronics are manufactured by LI-COR(slightly modi® ed from components used in Model 4000 DNA sequenc-er). The P/ACE 5000, Model 406 A/D converter, and System Goldsoftware are from Beckman Instruments.

FIG. 2. Drawing of P/ACE 5000 and NIR LIF detector interface. LIFmounting plate (1), self-aligning beam probe assembly (2), asphericcondenser lens (3), circular aperture (4), laser excitation ® ber (5), andLIF capillary cartridge (6).

factured by LI-COR Inc. was selected as the NIR LIFdetector. The laser and detector system are comparable tocomponents used in an automated DNA sequencing in-strument11,19 (LI-COR Model 4000), with the exceptionof modi® ed focal length optics and an extra bandpass® lter. The laser assembly contains a GaAlAs laser diodeemitting around 787 nm (20 mW peak power, modulatedwith a 50% duty cycle) and a focusing lens ( f 5 46 mm).The detector consists of a three-stage Peltier-cooled av-alanche photodiode. The detector assembly contains aplano-convex lens ( f 5 31 mm) to collect the ¯ uores-cence image, three identical bandpass ® lters (825 6 15nm) to reduce background noise from laser scatter (Ray-leigh) light, and a second plano-convex lens ( f 5 31 mm)to refocus the signal onto the APD photo-active area (0.5mm diameter). The APD signal is demodulated by a lock-in ampli® er and ® ltered prior to its output in analog form.The laser, microscope assembly, and detector electronicswere received as a gift from LI-COR Inc.

A Beckman Model 406 A/D converter was purchasedto collect the analog detector signal and convert it to adigital output for collection by the chromatographic soft-ware. A block diagram of the main instrument compo-nents is shown in Fig. 1.

Interface Design. The P/ACE 5000 uses a capillarycartridge system that allows for liquid cooling of the cap-illary and quick exchange between cartridges. The LIFcartridge contains an ellipsoidal mirror behind the capil-lary for ¯ uorescence emission collection at 180 8 from theangle of excitation. The mirror’ s centered opening pre-vents re¯ ection of the majority of the laser incidencelight. Laser output is delivered to the capillary via a ® ber-optic cable terminating in a self-aligning beam probe as-sembly. The LIF capillary cartridge, ® ber-optic cable, andself-aligning beam probe assembly are used unchangedfrom the Beckman LIF design. A more detailed descrip-tion and ® gure of this design can be found elsewhere. 20,21

The Beckman LIF cartridge mirror collects the ¯ uo-rescence signal and emits it as a collimated beam of lightat 180 8 from the angle of excitation. An aspheric con-denser lens (8.5 mm focal length and 12 mm diameter,

Melles Griot #01LAG000/066) is mounted immediatelypast the beam probe assembly to refocus the ¯ uorescenceemission to the appropriate object size for the LI-CORdetector. The distance between the LIF mirror and aspher-ic condenser lens was kept as short as possible, dictatedby physical limitations. An 800 m m circular aperture isinstalled at the focal point of the condenser lens. The NIRdetector is ® xed to an X-Y-Z micrometer stage installedon a removable CE mounting plate and focused onto theimage produced by the interface lens. A drawing of theinterface design is shown in Fig. 2. The diode laser isdisconnected from the microscope assembly and ismounted onto another X-Y-Z micrometer stage mountedon the side of the CE base. The laser signal is focuseddirectly onto the excitation ® ber, resulting in a 4 mWaverage excitation power at the capillary interface. Theoptical path of the complete system is shown in Fig. 3.

The analog detector output is connected to a Beckman406 A/D converter and collected by the chromatographicsoftware. The maximum detector output is matched to the2 V input limit of the A/D converter by using a simplevoltage divider circuit consisting of a 1 K V and 6 K Vresistor (output 5 input/7).

Detector Evaluation. The image size produced by theaspheric condenser lens is evaluated experimentally. Animage is obtained by varying the detector Y and Z po-sitions, respectively, from the point of maximum signalat the ® xed focal length distance. A 5 nM solution of IR-780 in methanol is continuously rinsed through a 75 m mi.d. capillary for the image mapping experiment. Detectorresponse is recorded and normalized to a percentage ofthe maximum signal. The detector distance required toreach 10% of signal on each side of the maximum, minusthe diameter of the detector active area (0.5 mm), pro-vides an estimate of the image diameter.

System sensitivity is evaluated by injecting decreasingconcentrations of a near-infrared dye, NN382. A 5.5 310 2 7 M stock solution is prepared in water and used the

APPLIED SPECTROSCOPY 1621

FIG. 3. Optical path of NIR LIF interface with CE instrument. Thediode laser output is focused into a ® ber-optic cable terminating in aself-aligning beam probe assembly. The beam probe assembly alignsitself with the LIF capillary cartridge and positions the laser excitationbeam onto the capillary window. Fluorescence emission is collected bythe mirror and re¯ ected as a collimated beam at 180 8 from the angle ofexcitation. The collimated beam is focused by the aspheric condenserlens to the appropriate image size, at the focal length of the detector.The detector optics ® lter the ¯ uorescence signal through three bandpass® lters prior to focusing the signal onto the window of the APD detector.

FIG. 4. Electropherogram of a 70 nL injection of 5.5 3 10 2 12 MNN382 (385 zeptomoles injected). Run is conducted at 21 kV on a barefused-silica capillary (57 cm 3 75 m m i.d., 50 cm to detector) with a50 mM phosphate run buffer (pH 7.2).

same day. The stock solution is diluted with 2.5 mMborate buffer (pH 8.0) to concentrations ranging between1.3 3 10 2 9 and 1.1 3 10 2 12 M. All dilutions from thestock solution are prepared in Nalgene to reduce adsorp-tion problems encountered in glass. Samples are injectedhydrodynamically by pressurizing the sample vial withnitrogen at 0.5 psi for a 15 s duration. The run bufferconsists of 50 mM sodium phosphate, pH 7.2, preparedby mixing equimolar solutions of sodium phosphatemonobasic and sodium phosphate dibasic until the properpH is reached. The ® nal buffer solution is ® ltered througha 0.45 m m nylon membrane followed by a 1 min ultra-sonication step to remove dissolved oxygen. The barefused-silica capillary (57 cm 3 75 m m i.d.) is rinsed suc-cessively with 0.15 N NaOH, water, and run buffer for 1min intervals prior to sample injection. The run is con-ducted in a normal polarity mode at 21 kV for 11 min.The detector voltage is maximized at 10 V with a gainsetting of 0.16 and 15 Hz frequency response. The datacollection rate of the A/D converter is 2 Hz.

RESULTS AND DISCUSSION

Selection of the aspheric condenser lens is an impor-tant part of the detector interface. Improper matching ofthe image size to the photodiode active surface area canhave a detrimental effect on detector performance. Over-® lling of the detector active surface area leads to a lossof signal, resulting in higher limits of detection. The ex-perimentally determined mean diameter of the image is0.575 mm, indicating a slight over-® lling of the 0.5 mmdetector window. On the basis of an area comparison,this result indicates a signal loss of about 30%. The actuallight loss is probably less, since spot intensity is nonuni-form and generally decreases away from the center. Therapid change in signal upon minor detector position

changes is also a good indication of an acceptable imagesize match.

System sensitivity was evaluated by injecting serialdilutions of the NN382 labeling dye. This dye has ab-sorption and emission characteristics (about 780 and 800nm, respectively) suitable for use with the system’s ex-citation and detection wavelengths. Furthermore, NN382is water-soluble and contains an isothiocyanate function-ality for labeling of primary amine groups. The structureand photochemical properties of this dye have been dis-cussed elsewhere. 22,23 A linear detector response (R 50.9999) was observed over more than a 250-fold rangebetween 1.4 3 10 2 9 and 5.5 3 10 2 12 M. The signal-to-noise ratio [SNR, signal/root mean squared (rms) noise]was 15 for a 70 nL injection of the 5.5 3 10 2 12 M so-lution. This value indicates a limit of detection (SNR 53) of approximately 80 zeptomoles. The electrophero-gram of a 70 nL injection of the 5.5 pM solution (385zeptomoles injected) is shown in Fig. 4.

The above-mentioned sensitivity results were obtainedunder 100% aqueous conditions. Additional improve-ments can be expected with the use of organic modi® ers,cyclodextrins, or micellar additives above their criticalmicelle concentration in the run buffer, from an improved¯ uorescence quantum yield when the dye is exposed toa more hydrophobic environment. The ability to use thesemodi® ers in the CE run buffer will depend on the speci® capplications for which the labeling dye is to be utilized.

The PMT-based Beckman LIF detector lists a signal-to-noise (signal/peak-to-peak noise) speci® cation of $ 10for a 1.8 attomole (10 2 18) injection of ¯ uorescein (520nm) and an 18 attomole injection of Cy5 (675 nm). Thisvalue corresponds to an SNR speci® cation (signal/rmsnoise) $ 50 for the above-mentioned injection amounts,assuming the rms noise is about 1/5 of the peak-to-peaknoise. The sensitivity of this detector decreases anotherorder of magnitude at 820 nm, as the quantum ef® ciency(QE) of the PMT drops from about 3% at 675 nm to, 0.3% at 820 nm.24 The improvement in sensitivity ofthe APD LIF system at 820 nm, with the use of NN382,can be estimated as follows:

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18 amol (Beckman LIF, SNR $ 50)

1.3 amol (NIR-LIF, SNR 5 50)

3% QE at 675 nm3 5 139-fold improvement.

0.3% at 820 nm

A more accurate estimate of sensitivity improvementof the APD vs. the PMT detector must take into accountdifferences in both excitation power and dye properties.The magnitude of the ¯ uorescence signal at low dye con-centrations is proportional to25

F 5 K f I0Îbc (1)

where K is a constant, I0 is the excitation power, f is the¯ uorescence quantum yield, Î is the molar absorptivity atthe excitation wavelength, and b and c represent cellpathlength and dye concentration, respectively. The ex-citation power listed for the Beckman LIF system is 3mW at 635 nm, while the NIR LIF system uses 4 mWat 787 nm. Cy526 has a higher Îmax and f than NN382 27

(Îmax 5 250 000 vs. 179 000; f 5 0.28 vs. 0.09). Since

Î is wavelength dependent, values were determined ex-perimentally for the wavelength of excitation by com-paring absorbance values at the maximum wavelength tothose at the wavelength of excitation. The calculated val-ues were 162 000 and 153 000 L cm 2 1 mol 2 1 for Cy5 (at635 nm) and NN382 (at 787 nm), respectively. SolvingEq. 1 using the appropriate laser excitation power, Î, andf for both systems shows a 2.5-fold increase in signalwith the use of Cy5 vs. NN382. This observation sug-gests a 340-fold sensitivity improvement for the APDover the PMT detector, at 820 nm, when one is usingdyes with comparable Î and f values.

The NIR LIF CE system has been used without prob-lems for about six months with over 1000 injectionsmade. Proper alignment of the optical path has been ver-i® ed on several occasions, and no signi® cant adjustmentshave been required. Alignment of the aspheric condenserlens mount with the beam block assembly is performedwith the aspheric lens removed, by inserting a stainlesssteel rod through the mount into the beam block. Theaspheric lens is then re-inserted into the self-centeringlens mount. Proper alignment of the laser with the ® ber-optic cable, and the detector with the interface lens, con-sists of performing a capillary rinse with a dilute dyesolution while adjusting the laser and detector micrometermounts for maximum signal.

CONCLUSION

A simple and reliable interface was designed for anavalanche photodiode-based, laser-induced ¯ uorescencedetector and a commercial automated capillary electro-phoresis instrument. The APD LIF system was optimizedfor use in the near-infrared region where detector sensi-tivity was improved about 340-fold compared to resultsfor a commercial PMT-based LIF detector. The NIR sys-tem uses more compact, rugged, and less expensive com-ponents, with an expected lifetime greater than 100 000hours. The fully automated injection, separation, and datacollection capabilities of the commercial CE system werepreserved. The NIR LIF system is used to evaluate theuse of near-infrared ¯ uorescent labels in analytical appli-cations.

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