S emicon ductor Laser Spectrometry

329ANALYTICAL SCIENCES JUNE 1993, VOL. 9

Reviews

S emicon ductor Laser Spectrometry

Totaro IMASAKA

Faculty of Engineering, Kyushu University, Hakozaki, Fukuoka 812, Japan

A semiconductor laser is more compact and less expensive than a conventional laser. Therefore, scientists prefer to use

this light source in analytical spectroscopy. However, the emitting wavelength of the semiconductor laser is located in

the near-infrared or deep-red region. Only a limited number of compounds are fluorescent in these spectral regions; we

have already developed many analytical reagents and procedures for application. Inorganic species such as potassium

ions and oxygen gas, organic molecules such as dyes and surfactants, and biological molecules such as metabolites,

enzymes, proteins, and deoxyribonucleic acid have been determined by this method. For example, amino acids are

determined at zeptomole levels after labeling them with a dye fluorescent in the deep-red region and separation by capillary

electrophoresis. Thus semiconductor laser spectrometry is advantageous for practical trace analysis.

Keywords Semiconductor laser, near-infrared spectrometry, laser spectrometry, photoacoustic spectrometry, absorp-

tion spectrometry, thermal lens spectrometry, fluorometry, practical trace analysis, optical fiber sensor,

liquid chromatography, capillary electrophoresis, micellar electrokinetic chromatography

1 Introduction

A laser is suitable as a light source in spectrometry, due

to its good beam coherence and monochromaticity. In

the beginning of the 1970's, the author was interested in

ultratrace analysis of organic compounds by using a

nitrogen-laser-pumped dye laser. This instrument can

easily be constructed in the laboratory and is chosen for

spectrometric works because of its wide tunability. When it was applied to fluorescence spectrometry, the

detection limit could be improved to 0.02 ppt for

fluorescein, which was more than three orders of

magnitude better than that obtained by a conventional fluorescence spectrophotometer with a xenon arc lamp.'

However, the present detection limit could be obtained

only for a strongly fluorescent compound dissolved in

ultrapure water. Thus this technique was, unfortunate-

ly, not applicable to a real sample containing many

chemical species. Fluorescence spectrometry is known to have high

selectivity, as given by optimization of both the excita-tion and fluorescence wavelengths. This is a striking

contrast to absorption spectrometry. However, additional

selectivity is required for application of this method to

practical trace analysis. So, we constructed an atmo-spheric-pressure nitrogen laser with a subnanosecond

pulse width and used it as a pumping source for a dye laser.2-4 Tunable subnanosecond laser pulses generated

were used for excitation of the sample in time-resolved

1 Introduction

2 Photoacoustic Spectrometry

3 Absorption Spectrometry

3.1 Phosphorus

3.2 Protein

4 Thermal Lens Spectrometry

4.1 Phosphorus

4.2 Iron

5 Fluorometry

5.1 Near-infrared fluorometry

5.1.1 Organic dye

5•l•2 Surfactant

5.l•3 Enzyme assay

5•l•4 Enzyme immunoassay

5.1.5 High-performance liquid

chromatography

5.2 Visible fluorometry using second

harmonic emission

329

330

331

331

332

5.2.1 Optical fiber sensor

5.2.2 Neutralization titration

5.2.3 Capillary electrophoresis

5.2.4 Micellar electrokinetic

chromatography

5.3 Visible fluorometry in deep-red

region

5.3.1 Organic compound

5.3.2 Surfactant

5.3.3 Protein

5.3.4 Enzyme assay

5.3.5 Deoxyribonucleic acid

5.3.6 Optical fiber sensor

5.3.7 Capillary electrophoresis

5.3.8 Micellar electrokinetic

chromatography

6 Future Studies 343

330 ANALYTICAL SCIENCES JUNE 1993, VOL. 9

fluorometry. This technique provided us additional selectivity for spectrometric analysis. In fact, selective detection of a specific compound with a long fluorescence lifetime, e.g. a polycyclic aromatic hydrocarbon, was

possible by temporal discrimination of the compounds with short fluorescence lifetimes.5 However, most organ-ic compounds have lifetimes of several nanoseconds, so that it was difficult to resolve these compounds on the fluorescence decay curve. Thus further improvement of selectivity was necessary for practical applications.

Time-resolved laser fluorometry was combined with high-performance liquid chromatography (HPLC) to overcome this problem.6 In this approach, the sample separated by a column could be sensitively detected by temporal discrimination of unwanted fluorescence from impurities. Moreover, the sample molecule was assigned from the fluorescence lifetime as well as from the retention time.' This method was applied to the sample extracted from airborne particulates, and several poly-cyclic aromatic hydrocarbons such as benzo[a]pyrene and benzo[ghi]perylene were assigned. Furthermore,

protein labeled with a hydrophobic fluorescence probe of 1-anilino-8-naphthalene sulfonate (ANS) was separated by HPLC, and hydrophobicity of protein was evaluated from the fluorescence lifetime since it increased with increasing the hydrophobicity.$ However, the detection limit obtained was subpicogram levels (210 fg for

perylene), while the value reported by a continuous wave (CW) laser, e.g. an argon ion laser or a helium cadmium laser was reported to be several femtograms. Pulsed laser fluorometry is considered to be more sensitive than CW laser fluorometry, due to background reduction by temporal discrimination. However, actual detectability was poorer due to pulse-to-pulse instability (20 - 50%) of the laser pulse. On the other hand, the background signal in CW laser fluorometry can be effectively sub-tracted by stabilization of the output power (<0.1%). Moreover, a flow cell was easily damaged by a laser pulse. A highly-repetitive laser with a small pulse energy, e.g. a mode-locked dye laser or a copper-vapor laser, is needed, but tunability and cost performance become poor. Thus a CW laser was more advantageous for trace analysis. Such analytical works had already been per-formed by many researchers. We then stopped working in this analytical field for several years.

In 1982, I was reading a magazine, in which I found an advertisement introducing a commercial semiconductor laser. This semiconductor laser was compact like a transistor and looked very practical as a light source in analytical spectroscopy. Moreover, a photodiode was contained in a package for monitoring the output power, which was useful to stabilize the output power by a feedback control. The output power of the semi-conductor laser was only several milliwatts, but ultra-trace analysis had been performed by using a laser operated at this power level. Unfortunately, the emitting wavelength was specified to 820 nm, which was too long in most analytical applications. I looked for a semi-conductor laser whose emitting wavelength was much

shorter than 800 nm to apply it to visible or ultraviolet

spectrometry. However, I noticed that no such semi-

conductor laser had been manufactured and that such a

laser was essentially impossible due to the band gap of the

semiconductor used. I looked for analytical reagents

and procedures which would be applicable to semi-

conductor laser spectrometry. However, I also noticed

that very few spectrometric works had been performed in

the near-infrared region. For example, some com-

plexes such as molybdophosphate had an absorption band at around 800 nm and were used for determination

of phosphorus, as described in the next chapter. On the

other hand, no organic compound was fluorescent in this

wavelength region except polymethine dyes, which were used as laser dyes emitting at above 700 nm. Unfortu-

nately, no analytical spectrometry had been reported by

using such near-infrared dyes.

I knew nothing about near-infrared spectrometry at

that time, but I thought a semiconductor laser was

essential to open a new field in practical trace analysis.

As introduced in this review, most papers related to

semiconductor laser spectrometry have been published

by our research group. However, many researchers

have started their investigations in this field and

submitted a lot of papers in international conferences.

This trend shows the potential of this method to be used

in practical trace analysis in the near future.

I have already published several review articles

concerned with semiconductor laser spectrometry.9l12

However, I would like to disclose the whole history in the

development of semiconductor laser spectrometry in this

review. I have taken care to prepare this manuscript so

as to describe disadvantages as well as advantages of this

method. So, I hope this critical review provides a

suitable introduction for researchers who are interested in starting practical trace analysis based on semi-

conductor laser spectrometry.

2 Photoacoustic Spectrometry13

1982, we tried to demonstrate the advantage of a semiconductor laser in spectrometric analysis. We chose determination of phosphorus by the Molybdenum Blue method, since its trace analysis was strongly requested for assessment of environmental water. The analytical procedure was well-known. The complex formed was measured by absorption spectrometry at around 800 nm. In this study, we decided to use photoacoustic spectrometry (PAS) for sensitive detection. Figure 1 shows the analytical instrument constructed. A semiconductor laser emitting at 818 nm

(5 mW) is modulated at 200 Hz and is focused into a sample cell. The piezoelectric transducer is installed in the cell for measurement of sound induced by a

photoacoustic effect, followed by light absorption and succeeding heat generation. The sample was mixed with a molybdenum solution according to a standard

protocol. The analytical curve was straight from 0 to


100 ppb of phosphorus, and the detection limit was

10 ppb. Thus we could demonstrate semiconductor

laser spectrometry, but the detection limit was much

poorer than that obtained by conventional absorption spectrometry. Such poor sensitivity is ascribed partly

to our lack of knowledge about photoacoustic spectrom-

etry and partly to the limited output power of the laser.

In fact, we had to operate the semiconductor laser above

the specification given by the manufacturer to get the

photoacoustic signal and thus destroyed several laser diodes. Nevertheless, we could publish the first report

of semiconductor laser spectrometry.

3 Absorption Spectrometry

3.1 Phosphorusl4 Due to poor sensitivity in photoacoustic spectrometry,

we tried to use a light emitting diode (814 nm) in absorp-tion spectrometry. This non-lasing devise has poor beam focusing capability but has a more stable and larger output power (36 mW). Then, it is better for use in absorption spectrometry. In this experiment, we used a dual beam configuration to cancel the variation of the output power. The detection limit of phosphorus could be improved to 15 ppt, corresponding to an absorbance of 1.5X106: It is noted that this detection limit is simply calculated from the signal-to-noise ratio of the back-

ground signal. For practical trace analysis, the back-ground signal should be minimized, since it varies if there are slight changes in color development of the sample and or if the cuvette is replaced.

3.2 Protein15 Protein is frequently measured by staining it with

Coumassie Brilliant Blue (or less recently with Methylene Blue), after separation by slab gel electrophoresis. At

present, a semiconductor laser emitting at 670 nm is commercially available, and this wavelength coincides with the absorption maximum of Methylene Blue. In the preliminary work, we tried to use fluorescence spectrometry because of its higher sensitivity, but no

fluorescence could be observed from the blue spot at

which Methylene Blue combined with protein is deposited. Later, we noticed that fluorescence of

Methylene Blue is completely quenched by polyacryl-

amide used for separation in slab gel electrophoresis.

Then, we detected the complex by absorption spectrom-

etry. Several proteins, such as macroglobulin,

phosphorylase b, glutamate dehydrogenase, lactate dehydrogenase, and trypsin inhibitor, were separated

and detected by this method. However, the base-line

noise was unexpectedly large, probably due to optical

inhomogeneity of the gel and the small beam size of the

laser beam. Thus good beam focusing capability was

not advantageously used in the present application, since

the separation resolution was not determined by the spot

size of the beam but determined by the resolution of the

gel plate used. However, it is noted that a semi-conductor laser is simpler and less expensive than the

light system consisting of a conventional electric bulb and

a dispersing element such as a grating or an interference

filter.

4 Thermal Lens Spectrometry

In order to improve sensitivity, we used thermal lens

spectrometry instead of conventional absorption

spectrometry.12,16,17 Figure 2 shows thermal lens

spectrometers based on single-beam and dual-beam

configurations. In the dual beam experiment, a He-Ne

Fig. 1

etry.

Experimental apparatus for photoacoustic spectrom-

Fig. 2 Block diagram of thermal lens spectrometer: (A) single beam system, (B) dual beam system.


laser is used as a probe beam, but it is possible to replace

it with another semiconductor laser emitting at a

different wavelength.

4.1 Phosphorus'8 Phosphorus was determined after the color

development described before. The detection limit and the enhancement factor, defined as a relative sensitivity of this method in comparison with conventional absorp-tion spectrometry, are summarized in Table 1. The enhancement factor given for aqueous sample solution is rather small, but it is greatly improved by measuring the sample after extraction into 2-butanol; the enhancement factor is 20 - 40 times increased for the sample in organic solvents.l5-17 The detection limits obtained by using solvent extraction are 0.2 ppb, which are mainly determined by the background signal from the solvent used. When molybdophosphate is extracted as an ion

pair into chloroform with a bulky cation such as Zephiramine, the enhancement factor was improved to 23 (theoretical value, 61) and the background signal occurring from the solvent could be reduced substantially. However, the background signal from the reagent blank was, unfortunately, not negligible in this case.

4.2 Iron19 Some heavy metals form charge transfer complexes

which have absorption bands in the near-infrared region. A typical example is a complex of Fell ion and 2-nitroso-5-diethylaminophenol. This complex has a broad absorption band at around 755 nm (e=4.2X 104), so a near-infrared semiconductor laser can be used for excitation. The enhancement factor obtained is summarized in Table 2. In this experiment, we noticed that aberration of the focusing lens seriously degraded the enhancement factor. A semiconductor laser has a large divergence angle due to a small active area, and a thick lens must be used to collimate the beam. An objective lens for a microscope provided the largest

optical transmission (78%) and the largest enhancement factor (90% of the theoretical value), while only low transmission (47%) and a small theoretical enhancement factor (11%) were obtained by a ball lens. It is apparent that thermal lens spectrometry provides a more sensitive analytical means than conventional absorption spectrometry. However, the background signal also increased even when impurities were carefully removed.

The thermal lens signal was measured quantitatively for many solvents. The result is shown in Table 3. The background absorption, which probably originates from overtone vibrations of the solvent molecule, is quite large for a molecule containing OH and CH bonds. This is due to the fact that such bonds have high vibrational energies and so the overtone vibrational bands extend to the near-infrared region. On the other hand, the background signal is small for a molecule containing only heavy atoms, e.g. CCl4 or CS2. The overtone band for C-Cl or C=S bond might be located in the infrared region, and the background signal is negligible in the near-infrared region. Unfortunately, most complexes are polar and are extracted into a polar solvent, though many exceptional cases are reported. Therefore, there are few successful examples in which semiconductor laser thermal lens spectrometry can be advantageously used.

Additional selectivity is really needed to overcome the

present problem, namely background reduction, in absorption-based spectrometry such as photoacoustic spectrometry or thermal lens spectrometry. However, no approach applicable to trace analysis by means of semiconductor laser spectrometry has been found yet, to our best knowledge, for a molecular sample in a condensed phase. Contrarily, a semiconductor laser with a narrow linewidth has been advantageously used in atomic absorption and fluorescence spectrometry. Thus a semiconductor laser has a potential to be used instead of a hollow cathode lamp, which may allow background subtraction by modulation of the laser

,21 frequency.21

5 Fluorometry

In fluorometry, the signal intensity is proportional to

the intensity of the exciting source. Thus a high-power

Table 1 Enhancement factor (E) and detection limit (DL) in determination of phosphorus (ppb)

Table 3 Light absorption by solvents in near-infrared region


light source is essential to increase the sensitivity. In

most fluorometric works, the detection limit is

determined by the intensity of the scattered light from the

exciting source or the background signal from impurity

fluorescence. The laser is monochromatic and is well-

collimated, and so the scattered light can be efficiently

reduced by spectral and spatial filters. Thus a laser is

suitable as a light source in fluorometry. Furthermore,

the maximum signal intensity is obtained only when the

excitation and fluorescence wavelengths are optimized,

as described. Thus this spectrometric method has

higher selectivity than absorption spectrometry. The

background signal from impurities decreases with

increasing the excitation and fluorescence wavelengths.

Thus ultratrace analysis may be established by near-infrared semiconductor laser fluorometry. Light

absorption by overtone vibration of the solvent molecule,

which is a serious problem in absorption spectrometry,

gives no background fluorescence. Thus a semiconduc-tor laser may provide a useful means for practical trace

analysis.

5.1 Near-infrared fluorometry 5. 1.1 Organic dye22-2a

A semiconductor laser fluorometer, shown in Fig. 3, was constructed and used for detection of a polymethine dye, 3,3'-diethyl-2,2'-(4,5,4',5'-dibenzo)thiatricarbocya-nine iodide (DDTC), dissolved in methanol. The chemical structure is shown in Fig. 4. The detection limits obtained by using a semiconductor laser (786 nm, 5 mW) were 5X101' M for a lock-in amplifier system and 5X10-12 M for a single-photon counting system. These values are much better than a value of 3X101° M

obtained by a conventional fluorescence spectrometer. As expected, the background signal was negligibly small. Then, the detection limit will be improved further by using a semiconductor laser with a larger output power.

A laser has good beam focusing capability and

provides its best performance when the exciting beam is focused tightly to the sample localized in a small volume. A micro fluorescence detector consisting of a hollow capillary and an optical fiber was combined with semiconductor laser fluorometry. A 60-n1 sample was introduced into a capillary by a micro valve injector. In this experiment, two optical configurations, shown in Fig. 5, were used; an optical fiber (core diameter 20 µm, cladding diameter 150 µm) inserted into a hollow capillary (200 µm i.d.) is used for introduction of the exciting beam or detection of fluorescence. The detection limits obtained were 12 fg and 90 fg for these two configurations. It is noted that such high sensitivity is achieved even though a low-power semiconductor laser

(3 mW) was used. When a semiconductor laser is driven by a short pulse

current, a picosecond pulse (ca. 100 ps) can be generated. By a combination with a time-correlated photon counting system, a fluorescence lifetime was measured with a time resolution of 480 ps. The fluorescence lifetime of DDTC depended on the dielectric constant of the solvent. Then, the hydrophobicity of a specific binding site attached to this molecule was evaluated. The time resolution was determined by a transit-time-spread of the photomultiplier. This value might be substantially improved by using a streak camera instead

Fig. 3 Experimental apparatus for fluorometry using near-

infrared semiconductor laser.

Fig. 5 Schematic diagram of experimental apparatus: (1) sample excited through optical fiber, (2) fluorescence

detected through optical fiber.


of a time-correlated photon counting system. 5.1.2 Surfactant22

The number of organic compounds fluorescent in the near-infrared region is quite limited. Then, an analyt-ical sample must be measured after some analytical

procedure or treatment. A surfactant is well-known to be measured by fluorometry after solvent extraction with a fluorescent dye. A polymethine dye has a positive charge and forms an ion pair with a negatively charged surfactant. For example, sodium lauryl sulfate (SDS) was extracted with DDTC into benzene and was measured by semiconductor laser fluorometry. The detection limit was 1X107 -M, and the blank fluorescence corresponded to 3X10-7 M SDS. A similar result was obtained for sodium dodecyl benzenesulfonate (DBS). The present detection limit was similar to or slightly better than the value obtained by a conventional method. 5 Enzyme assay25

In order to measure a biological molecule, it is necessary to develop a fluorescent reagent reactive to a specified molecule. However, no such chemical reagent fluorescent in the near-infrared region had been reported; such reagents have been synthesized until now and are reviewed elsewhere.26 Then, we mixed Indocyanine Green, a soluble and stable polymethine dye in aqueous solution, with many chemicals. The chemical structure of Indocyanine Green is shown in Fig. 6. We found that hydrogen peroxide (H2O2) strongly quenches fluo-rescence of Indocyanine Green. Then, we tried to use this near-infrared fluorescent dye for monitoring an enzyme reaction producing hydrogen peroxide. For example, xanthine is known to be converted to uric acid by xanthine oxidase as follows:

xanthine Xanthine+ 02 + H2O oxidase uric acid + H2O2. (1)

Then, the concentration of xanthine may be deter-

mined by measuring fluorescence quenching of

Indocyanine Green.

In the preliminary study, this attempt was successful,

but later we could not reproduce this enzyme reaction. We were puzzled for a long time. However, we noticed

that impurities in the water played an important role. We used water purified by a metal distiller in the

preliminary study and used ultrapure water further

purified by a glass distiller and deionized by a ion-exchange column in later works. A reproducible result could be obtained only when water purified by the metal distiller was used in the experiment. An impurity such as a heavy metal might be contained in water, and it

probably acted as a catalyst to convert hydrogen peroxide (H2O2) into hydrogen oxide radical (OH), which is reactive to Indocyanine Green. Then, we investigated the effect of a heavy metal systematically, and found that the presence of Fe" ion is essential to obtain a reproducible result. Figure 7 shows the effect of hydrogen peroxide on the fluorescence intensity of Indocyanine Green in the presence of Fe" ion. The fluorescence intensity of Indocyanine Green is strongly

quenched by hydrogen peroxide. By using this chemi-cal reaction, the calibration curve was constructed from 5X105 to 5X107 M for xanthine. The detection limit was 10-' M level. This chemical reaction was also used for measurement of activity of xanthine oxidase. 5 . 1 . 4 Enzyme immunoassay27

In the previous section, we assumed that an OH radical attacks Indocyanine Green to form a radical at the

polymethine chain, producing a stable dimer. This reaction cleaves a conjugated bond, and then the absorption band might be shifted from the near-infrared to the visible region. It is known that peroxidase

produces an OH radical. Then, it may be used for monitoring the enzyme reaction using peroxidase. In order to confirm this idea, we added peroxidase into the aqueous solution containing Indocyanine Green and hydrogen peroxide. An analytical curve was construct-Fig. 6 Chemical structure of Indocyanine Green.

Fig. 7 Fluorescence spectra for Indocyanine Green measured after reaction with hydrogen peroxide at concentrations

indicated on curves. Spectra were measured after 3 h (left) and 4 h (right).


ed from 0 to 400 µunits/ml, and the detection limit was 40 µunits / ml.

For selective determination of protein, an immuno-logical reaction has been used in biological assay. One

possible scheme is drawn in Fig. 8. In sandwich enzyme immunoassay, enzyme-labeled antibody is attached to the antigen (sample) and its concentration is determined by measuring the enzyme activity; absorption changes are measured after addition of a substrate. We followed this protocol, but we used near-infrared semiconductor laser fluorometry and Indocyanine Green as a substrate. A calibration curve was constructed from 0 to 150

µunits/ ml, the detection limit being 10 µunits/ ml. 5.1.5 High-performance liquid chromatography2a,29

Many chemical species are included in a real sample, and they should be separated before detection. High-

performance liquid chromatography is used for this purpose, since it is applicable to polar and even non-volatile samples. In order to demonstrate the advantage of HPLC/ semiconductor laser fluorometry, we separated polymethine dyes by HPLC and detected them by a semiconductor laser fluorometer equipped with a flow cell. The chromatogram obtained is shown in Fig. 9. The eluent was a mixture of water and methanol (1: 9), containing 15 mM choline chloride [(2-hydroxyethyl)trimethylammonium chloride] which was added to form an ion-pair with the polymethine dyes. The detection limit obtained by semiconductor laser fluorometry was more than two orders of magnitude better than that obtained by a conventional fluorometric detector. High-performance liquid chromatography is currently used for determination of many biological substances. For samples with no absorption band in the visible or ultraviolet region, they should be labeled by a fluorescent

dye. However, no labeling dye applicable to near-

infrared fluorometry had been developed before. We

investigated which organic dye was reactive to protein.

Fortunately, we found a negatively-charged polymethine

dye, i.e. Indocyanine Green, is bound to protein

exceptionally. This compound is physically adsorbed

on the surface of protein and increases the fluorescence

intensity, as shown in Fig. 10, probably due to a

hydrophobic interaction. Thus this compound can be

used as a labeling reagent of protein. Figure 11 shows a

chromatogram of human serum, which is obtained by

labeling protein with Indocyanine Green and by separation with gel-filtration HPLC . Several proteins

Fig. 8 Procedure for enzyme immunoassay based on near-infrared semiconductor

laser fluorometry.

Fig. 9 Chromatogram for equimolar mixture of three poly-

methine dyes with semiconductor laser fluorometry for

detection. The numbers specified in the figure are com-

mercial code numbers given by Nippon Kanko Shikiso.


in human serum are clearly resolved. The detection

limit obtained by semiconductor laser fluorometry was

more than two orders of magnitude better than the values

obtained by conventional spectrometric detectors.

5.2 Visible fluorometry using second harmonic emis-

sion

Near-infrared fluorometry suffers from a shortage of

chemical reagents applicable to fluorometry. On the

other hand, there are many chemical reagents which can

be applied to fluorometry in the ultraviolet and visible

regions. If the emitting wavelength of the semiconduc-

tor laser is extended to 300 - 500 nm, it is possible to find many analytical applications. It is difficult to generate laser emission directly at shorter wavelengths, but it is

possible to convert the laser wavelength into such regions by a nonlinear optical effect, e.g. second harmonic

generation. For example, the emission wavelength of the near-infrared semiconductor laser can be frequency-doubled from 800 nm to 400 nm. Thus visible fluoro-metry can be demonstrated by using a frequency-doubled near-infrared semiconductor laser. 5.2.1 Optical fiber sensor3o

In the preliminary work, a fundamental beam of the semiconductor laser (820 nm, 20 mW) was focused into a nonlinear optical crystal of potassium dihydrogen

phosphate(KDP). The conversion efficiency was 2.5X106, and the output power obtained was 50 nW at 390 nm. The emission intensity was very small, but it was possible to visualize the laser beam spot by scattering it on a white piece of paper. This laser power was sufficient for use in fluorometric determination of organic molecules. In fact, a fluorescent dye, 7-diethylamino-4-methylcoumarin could be measured to 5X108 M.

An optical fiber has been developed for use in data communication. It transmits the signal with no appreciable loss for a long distance. This characteristic is desirable for a transmission line in a remote sensing system. Thus a sensing probe and a transmission line can be directly coupled as an optical fiber sensor. For example, a fluorescent material is attached to the top of the optical fiber. Changes in fluorescence characteris-tics, e.g. fluorescence intensity, spectral shape, or lifetime are monitored by introducing the exciting light and collecting the fluorescence through an optical fiber. For this purpose, it is necessary to use an exciting source with good beam focusing capability and good mono-chromaticity, since the core diameter of the optical fiber is small and the scattered light must be reduced by using an optical filter for measurement of weak fluorescence. A laser was an ideal light source, but its large dimension and expensiveness prevented its practical use.

Due to good beam focusing capability and good monochromaticity of the second harmonic emission of the semiconductor laser, it is advantageous for use in an optical fiber sensor system. Figure 12 shows a structure of the optical fiber sensor system constructed. In order to detect oxygen in the air, benzo[ghi]perylene was dissolved in silicone grease and was wrapped with a gas-

permeable membrane. This sensing head is attached to the top of the optical fibers. The concentration of oxygen was determined from 0 to 40% by dynamic fluorescence quenching of benzo[ghi]perylene. 5.2.2 Neutralization titration31

Due to good beam focusing capability, a laser is frequently used as a light source in nonlinear optical spectrometry. In our preliminary work, we tried to use a semiconductor laser as a light source in two-photon excitation fluorescence spectrometry. We tightly focused the beam by an objective lens for a microscope,

Fig. 10

Green.

Excitation and emission spectra for Indocyanine

Fig. 11 Chromatogram for human serum labeled with Indo-

cyanine Green.


since the fluorescence intensity is expected to be

proportional to the square of the radiation field. After some experimental checks, we could observe the fluorescence signal. However, the relative signal intensities for several organic dyes were different from those obtained by an excimer-laser-pumped dye laser with an output power of 1 MW. We were puzzled for a long time, since the relative intensity must not depend on the output power of the laser until signal saturation. After submission of a dissertation to the department by my undergraduate student, we were very surprised to notice that the fluorescence originated from excitation of the sample by the second harmonic emission generated by the semiconductor laser itself! As shown in Fig. 13, the semiconductor laser produced the second harmonic emission at picowatt levels directly from the laser diode, since it was also made of a nonlinear optical material. The conversion efficiency was 1.7X1011. The emission intensity was extremely weak, and it was difficult to confirm the laser beam spot by our naked eyes. However, it was still possible to use it as an exciting source in fluorometry. The analytical curve was con-structed for perylene, and the detection limit was 10-6 M.

The dependence of the fluorescence intensity on pH

was measured for 8-hydroxy-1,3,6-pyrenetrisulfonic acid

(HPTS), which was used as a fluorescence indicator. Since this molecule is fluorescent only in the alkaline solution, it can be used as a pH indicator for neutral-ization titration. This possibility was confirmed experi-mentally by using the present instrument.

Later, we noticed that this phenomenon of generating

the second harmonic emission in the laser diode itself had

been found by other researchers before our publication. However, the present report is, to our best knowledge,

the first publication of its analytical application.

5.2.3 Capillary electrophoresis32

Liquid chromatography is currently used for

separation of biological substances. Recently, capillary

electrophoresis has attracted much biochemists'

Fig. 12 (A) Optical fiber sensor system using second harmonic emission of semiconductor laser. (B) Sensing device for detection of oxygen.

Fig. 13 Dependence of output power on diode current.


attention, due to its better separation resolution. For example, a resolution of several hundred thousand theoretical plates is routinely achieved in free zone capillary electrophoresis. This value is further improved to a million in capillary gel electrophoresis. Moreover, the separation time is made rather short by application of a high electric potential. In most experiments, the sample is determined by absorption spectrometry. The path length is only 50 µm typically, so that the sensitivity is practically limited to a 10-5 M range. Fluorometry is useful for sensitive detection of the sample, but an exciting beam should be tightly focused into a capillary. Thus laser fluorometry is advantageous in this application. However, only one commercial instrument including a laser (Beckman Instruments) is so far available, due to the expensiveness of the laser.

Recently, a compact laser system emitting the second harmonic emission of the semiconductor laser has been commercialized. This laser, called a blue semiconduc-tor laser, consists of a near-infrared semiconductor laser and a waveguide made of a nonlinear optical crystal for frequency doubling. The conversion efficiency is

greatly improved by confining the laser beam in a narrow channel of the waveguide.

We constructed the laser fluorometric system shown in Fig. 14. The exciting source used is a blue semi-conductor laser (415 nm, 50 µW, linewidth 3 nm, beam divergence 1°, linearly polarized) consisting of a waveguide of LiNb03 for generation of the second harmonic emission, a near-infrared semiconductor laser

(830 nm, 40 mW), and a power supply (850 MHz). The body of the laser is only 8 cm long, 2 cm wide, and 2 cm high. There are many labeling reagents in the blue spectral region, e.g. fluorescein isothiocyanate or naphthalene dialdehyde. In our work, 7-diethylaminocoumarin-3-carboxylic acid succinimidyl ester (DCCS) was used for

labeling amino acids which were separated by capillary electrophoresis. The electropherogram obtained for a mixture sample containing four amino acids is shown iii Fig. 15. All the components are clearly resolved; the side reaction products are also detected and specified as "reagent" . The detection limit was 100 attomole levels for these amino acids. 5.2.4 Micellar electrokinetic chromatography33

Capillary electrophoresis has a high separation resolu-tion, but it can only be applied to charged molecules. Neutral species are separated by a similar technique, i.e. micellar electrokinetic chromatography. A surfactant is added in a buffer solution and the micelle which forms acts as a quasi-solid phase in chromatographic sepa-ration. In this approach, the solution moves as a plug flow, and then diffusion of the sample is minimum. This is in striking contrast to conventional chromatog-raphy. For separation of neutral molecules such as polycyclic aromatic hydrocarbons, y-cyclodextrin was added to the mobile phase to increase the solubility of the analyte. As shown in Fig. 16, 1-aminoanthracene and benzo-

[a]pyrene were separated and detected by micellar electrokinetic chromatography/ semiconductor laser fluorometry. The detection limit was femtogram levels, which is comparable to or slightly better than the best value obtained by HPLC/ conventional laser fluorome-try. The high mass sensitivity obtained here even by using a lower-power laser is ascribed to a smaller sample injection volume (1 nl), since the sample is more concentrated at the sample detection port.

The optimum power for capillary electrophoresis or capillary electrokinetic chromatography is known to be ca. 10 mW. Thus the sensitivity can be greatly im-

proved by increasing the output power of the laser. Until now, the output power of a frequency-doubled semiconductor laser has been improved to 40 mW in a research level, though a complicated temperature control

Fig. 14 Schematic diagram of experimental apparatus for

capillary zone electrophoresis combined with semiconductor

laser fluorometry.

Fig. 15 Electropherogram for visible dye (DCCS).

amino acids labeled with


system and a laser cavity to confine a laser beam are necessary. Moreover, a solid-state laser pumped by a high-power semiconductor laser is commercialized and has a large output power. For example, the frequency-doubled (532 nm), frequency-tripled (355 nm), or fre-

quency-quadrupled (266 nm) emission of a neodymium yttrium aluminum garnet (Nd : YAG) laser has sufficient output power; it will find many applications in the near future.

5.3 Visible fluorometry in deep-red region To our best knowledge, only a group of compounds,

namely polymethine dyes, are fluorescent in the near-

infrared region. These compounds dissolved in aqueous

solution are less stable under irradiation of the visible and ultraviolet light, since these are used as photosensitizing

dyes. So, we thought they were not suitable for practical

applications; some polymethine dyes are known to be

stable enough under near-infrared radiation. Further-

more, the dyes listed in a manufacturer's catalog had no

active site for combination with a biological molecule.

Recently, a semiconductor laser emitting at shorter

wavelength, e.g. 670 nm, was commercialized. In this

deep-red region, there are many fluorescent dyes

commercially available. Some of them have a sufficient

stability and are already used in analytical purposes.

Then, we started analytical spectroscopy using a 670-nm

semiconductor laser and these fluorescent dyes.

5.3.1 Organic dye34

In the preliminary work, we demonstrated detection of

Rhodamine 800, which was used as a laser dye. The absorption band is located at around 680 nm and the

lasing wavelength at around 800 nm. The detection

limit was 4X10'2 M, which is slightly better than that

obtained by near-infrared semiconductor laser

fluorometry. Chemical structures for several dyes

available in the deep-region are shown in Fig. 17.

5.3.2 Surfactant3s

As was done in near-infrared fluorometry, a surfactant

was measured by solvent extraction with a dye

fluorescent in the deep-red region. A surfactant, SDS,

was extracted with Rhodamine 800 into m-xylene, and

the fluorescence intensity of the ion-pair in the organic

phase was measured. The detection limit was 10-' M. The minimum detectability was similar to that obtained

by near-infrared fluorometry using a polymethine dye.

Present semiconductor laser fluorometry might be also

applied to the method specified by Japan Industrial

Standard, since Methylene Blue fluorescent in the deep-- red region is used as an analytical reagent .

5.3.3 Protein34

No chemical reagent has been developed which is

fluorescent in the deep-red region and is useful to label a

specific organic or biological molecule. However, some

organic dyes have an active site to combine with such

molecules. As shown in Fig. 18, the NH group in a dye

and the COOH group in protein can be combined with a

bifunctional reagent such as water-soluble carbodiimide.

The labeling efficiencies are summarized in Table 4.

Nile Blue is most efficiently bound to albumin, since it

Fig. 16 Chromatogram for and benzo[a]pyrene.

mixture of 1-aminoanthracene

Fig. 17

region.

Chemical structure of dyes fluorescent in deep-red


has the most reactive NH2 group. However, the fluorescence intensity measured by semiconductor laser fluorometry is rather small. This is ascribed to low excitation (11 %) and detection (31%) efficiencies, which are due to mismatches between the absorption maximum of the dye and the emitting wavelength of the semi-conductor laser. The efficiencies are better for Oxazine 750. Methylene Blue can be also used for labeling

protein by an electrostatic force. The detection limit of albumin injected was 2.5X10-9 M, corresponding to a mass detection limit of 0.25 pmol. This value was improved to 0.13 pmol for Oxazine 750. The labeling efficiency was very small in this study. The mole ratio of bound Methylene Blue to albumin was 0.04, which is much smaller than the number of the COOH sites (47) in albumin. This is attributed to the fact that the NH2

group directly coupled with a phenyl ring is not so reactive. 5.3.4 Enzyme assay36

Methylene Blue is converted to colorless Leucomethyl-ene Blue in the presence of diaphorase (DP) and reduced nicotinamide adenine dinucleotide (NADH). Thus an enzyme reaction producing NADH can be monitored by visible semiconductor laser spectrometry using Methylene Blue. This reaction scheme is shown in

Fig. 19. For example, lactate hydrogenase (LDH) is determined by

Lactic acid + NAD+ LDH P

yruvic acid + NADH + H+. (2)

A human serum sample containing 0.2 U/ml of LDH was easily detected by absorption spectrometry using a semiconductor laser. A flow system was used to reduce oxidation of Leucomethylene Blue by oxygen dissolved in the sample solution. On the other hand, ethanol can be measured by

Ethanol + NAD+ ADH A

cetaldehyde + NADH + H+. (3)

The detection limit of alcohol dehydrogenase (ADH) obtained by fluorometry was 4 mU, that of ethanol being 10 nmol. The concentration of ethanol in normal blood is ca. 2 µmol/ ml; the legal concentration limit for a driver in Japan is 10 µmol/ ml. Hence, this method has sufficient sensitivity for practical assay of ethanol.

The sample containing NAD+ was injected into a merging zone containing DP and Methylene Blue. Leucomethylene Blue partially coexisting in the Methylene Blue was oxidized by NAD+ to fluorescent Methylene Blue. The calibration graph was linear over the range 0 - 40 nmol, over which the signal increased 1.4 times. 5.3.5 Deoxyribonucleic acid3?

Methylene Blue is know to be intercalated into a double strand of deoxyribonucleic acid (DNA). The absorption band is shifted to shorter wavelengths and the fluorescence intensity decreases substantially, as shown in Fig. 20. Thus the concentration of DNA can be determined by measuring fluorescence quenching. The concentration of DNA could be determined to 10-5 M base-pair by this method. 5.3.6 Optical fiber sensor

A fiber-optic chemical sensor for potassium ions is constructed using a plasticized poly(vinyl chloride)

(PVC) membrane containing valinomycin. A schemat-ic diagram for measurement is shown in Fig. 21. A

Fig. 18 Binding reaction of

soluble carbodiimide.protein and dye using water-

Fig. 19 (A) Enzyme reaction scheme using Methylene Blue. (B) Structures of Methylene Blue (left) and Leucomethylene

Blue (right).

Table 4 Fluorescence intensity of labeled albumina

a.

b.

C.

Values are normalized to those for Methylene Blue.

Calculated from results obtained by lamp excitation.

Wavelength for fluorescence detection given in parentheses.


hydrophobic fluorescent dye of Oxazine 750 is covalently bound to PVC, as shown in Fig. 22. The fluorescence intensity from the PVC membrane is measured by using a visible semiconductor laser emitting at 680 nm. The sensor response was reversible to the potassium ion concentrations in the 10-3 -10-1 M range. The selec-tivity coefficient for sodium ion was 2X103. 5.3.7 Capillary electrophoresis32,39,40

In the previous chapter, a blue semiconductor laser, i.e. the second harmonic emission of the near-infrared semiconductor laser, was used as an exciting source for detection of the sample separated by capillary electrophoresis. However, the output power of the commercial blue laser is limited to 0.1 mW or less. Moreover, the cost of the blue semiconductor laser is similar to that of the capillary electrophoresis system. Then, a fundamental beam of the semiconductor laser must be used for practical application to capillary electrophoresis. Several forms of chlorophyll, e.g. chlorophyll a, are

known to be fluorescent in the deep-red region. Then, chlorophyll a was measured by using a 670-nm semi-conductor laser to check performance of the capillary electrophoresis system developed. A sharp peak was observed in an electropherogram, giving several hundred thousand theoretical plates.

Unfortunately, only a limited number of samples are fluorescent in the deep-red region. However, indirect fluorometry may be applied to overcome this problem; a fluorescent dye is flowed continuously and all the chemical species are measured as dip signals without using any labeling procedure. In this approach, adsorption of the dye on the capillary surface must be negligible. We injected a dye solution into the capillary and observed the peak shape. Unfortunately, the peak was broadened and had a long tail for all the compounds investigated, i. e. Methylene Blue, Oxazine 750, and Rhodamine 800. However, this adsorption could be minimized by using a capillary treated with an alkylsilane. Figure 23 shows an electropherogram observed for a mixture sample containing glycine and

proline by using Methylene Blue as a fluorescent dye.

Fig. 20 Absorption (1) and fluorescence (2) spectra of Methylene Blue in absence (A) and presence (B) of DNA.

Fig. 21

sensor.

Experimental apparatus of fiber-optic chemical

Fig. 22 Schematic illustration of PVC-bound Oxazine 750.


The detection limit was 1 picomole level in this approach. In indirect fluorometry, the compounds are measured

as dip signals from a base line, and therefore the

sensitivity is quite limited. To overcome this problem,

the chemical species of interest must be directly labeled

with a fluorescent dye. However, no such labeling reagent had been developed for use in capillary electrophoresis.

So we newly synthesized a labeling reagent according to

the procedure shown in Fig. 24. The compound

synthesized has a chromophore similar to Methylene

Blue and a succinimidyl ester to combine with an amino

group. Amino acids were labeled with this compound and were detected after separation by capillary

electrophoresis. The result is shown in Fig. 25. Sharp signal peaks, assigned to arginine and glycine, are

observed in the electropherogram; the strong signal scaled out at 6 min is due to the reagent or side-reaction

products which appeared in the synthesis procedure since the labeling reagent had not been purified in this study. The detection limit was 10 picomole levels.

More recently, we used a new labeling reagent, 9-cyano-N, N, N'-triethyl-N'-triethyl-N'-(5'-succinimidyl-oxycarbonylpentyl) pyronin, chloride (pyronin succini-midyl ester) synthesized for the present purpose. Moreover, a sheath flow detector was developed to minimize the background signal occurring from the light scatter at the cell wall. Five amino acids were clearly resolved and the detection limit could be improved to subattomole levels (800 zeptomole for glycine). Thus capillary electrophoresis/ semiconductor laser fluorome-try provides an ultrasensitive means for practical trace analysis. Protein was also labeled with the above fluorescent reagent and was measured after separation by capillary electrophoresis. The capillary was treated with an alkylsilane to reduce adsorption of protein onto the capillary surface by electrostatic forces. Furthermore, a surfactant of Brij 35 was added in the buffer solution to reduce adsorption. This method was applied to immunoglobulin G, trypsin, and xanthine oxidase. The peak was rather broad, due to a strong interaction between protein and the capillary surface. The detection limit was 600 attomole for xanthine oxidase. 5.3.8 Micellar electrokinetic chromatography41

As described in the previous chapter, neutral molecules can be separated by micellar electrokinetic chromatog-raphy. This technique is also used to change the separation mode, i.e. selectivity. We used a surfactant with a positive charge to modify the capillary surface; a silanol group with a negative charge at the capillary surface is bound to a surfactant with a positive charge and adsorption of Methylene Blue to the capillary surface is minimized. It is noted that an electroosmotic flow occurs toward a positive electrode and a negative

potential must be applied to the sample inlet side. In this approach, flavin adenine dinucleotide (FAD) and deoxyadenosine monophosphate (dAMP) could be re-solved and detected indirectly, based on semiconductor laser fluorescence detection. The detection limit was

Fig. 23 Indirect fluorometry of amino acids measured by

flowing Methylene Blue as fluorescence reagent.

Fig. 24 Scheme for organic synthesis of labeling reagent.


100 femtomole levels.

6 Future Studies

In 1960, a laser was invented by Maiman. Since then,

many types of laser and nonlinear optical phenomena

were discovered. However, we had to wait more than 20

years for their practical use, since no reliable laser appeared until the beginning of the 1980's. For example, we used a nitrogen-laser-pumped dye laser in

analytical spectroscopy, but we suffered from problems

related to maintenance of the nitrogen laser and

degradation of the laser dye. A manufacturer of the

analytical instrument says that a commercial instrument

must be operated continuously more than 1 year without any maintenance. Thus it was difficult to construct an

analytical instrument using a laser. From the latter half

of the 1980's, the situation was completely changed, since

a semiconductor laser was manufactured by mass

production technology for use in a compact disk and a barcode reader. The semiconductor laser is reliable and

can be operated continuously for more than several years

without any maintenance. Thus a semiconductor laser

promises to open a frontier in practical trace analysis. In the future, almost all the lasers constructed now will

be replaced with semiconductor lasers or related

products. For example, a solid-state laser pumped by a semiconductor laser has already frequency-quadrupled

and is used as a pumping source for an optical parametric

oscillator, which generates tunable laser emission in the

entire visible region and is further extended to the

ultraviolet region by further frequency doubling. If

necessary, the frequency of the semiconductor laser is locked to the wavelength of interest by using a waveguide

with a periodic refraction index change. Thus state-of-

the-art laser technology will allow many applications

requiring a high performance laser with a minimum cost.

Therefore, a semiconductor laser will completely change

the situation encountered now and will provide an

opportunity to construct a new analytical instrument for

many analytical chemists who are now using an

incoherent light source or even a conventional laser source.

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(Received January 8, 1993) (Accepted February 25, 1993)

S emicon ductor Laser Spectrometry

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