Anal. Chem. 1999, 71, 2262-2265
Diffractive Optical Chemical Sensor Based on Light Absorption Fumihiro Nakajima, Yasuyuki Hirakawa, Takashi Kaneta, and Totaro Imasaka*
Department of Chemical Systems and Engineering, Graduate School of Engineering, Kyushu University, Hakozaki, Fukuoka 812, Japan
A new type of chemical sensor based on light absorption is proposed. An array of zones alternatively containing the pH indicator thymolphthalein is formed in a gelatin film. By changing the sample solution from acidic to alkaline, a blue stripe appears in the gelatin film. This acts as a transmission grating and diffracts the introduced laser beam. Theory predicts that this method, which is based on light absorption/beam diffraction, is as sensitive as or more sensitive than fluorometry.
Absorption spectrometry is a versatile technique which is widely used in a variety of applications. In some applications, for example, trace analysis, the sensitivity of the technique is insufficient. On the other hand, fluorescence spectrometry is very sensitive, permitting the detection of even single molecules using state-of-the-art laser fluorometry. Unfortunately, only a limited number of organic compounds are fluorescent, which limits the application fields of fluorometry. It is possible to measure a nonfluorescent sample after labeling it with a fluorescent tag, but labeling efficiency is often poor and the tag determines the detection limit in practical trace analysis.1 Thus an analytical method that is directly applicable to nonfluorescent samples and is as sensitive as fluorometry would be highly useful. A variety of photothermal spectrometric techniques have been developed for the measurement of low levels of light absorption, but a high-power continuous-wave laser is needed for trace analysis.2,3 Unfortunately, the wavelength tunability of the laser is very limited, especially in the deep ultraviolet region. For these reasons, such methods have not yet been used in practical trace analysis. Recently, various types of diffractive optical elements (often referred to as diffractive optics) have been developed for use in a display. In this case, numerous lines are grooved on a plastic or glass plate for the purpose of beam diffraction. The simplest diffractive optical element is a one-dimensional grating. Such a diffractive optical element is used for the projection of various patterns (e.g., dots, lines, circle, cross, grid, and star), and even letters and figures can be projected on a large screen. * Corresponding author: (tel) 81-92-642-3563; (fax) 81-92-632-5209; (e-mail)
[email protected]. (1) Banks, P. R. Trends Anal. Chem. 1998, 17, 612-622. (2) Imasaka, T.; Ishibashi, N. Trends Anal. Chem. 1982, 1, 273-277. (3) Imasaka, T.; Ishibashi, N. Prog. Quantum Electron. 1990, 14, 131-249.
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In analytical spectroscopy, beam diffraction is used for the detection of changes in refractive index. This approach is in current use in immunoassays, in which a change in refractive index arising from the adsorption of a specific protein as the result of an immunological reaction is detected by measuring the intensity of the diffracted beam.4 Sample elution in capillary electrophoresis is also detected by beam diffraction, since the refractive index of the eluent containing the sample is slightly different from that of the solvent.5 This approach provides a universal detector which can be applied to colorless samples. However, it is neither specific nor sensitive and is difficult to apply to trace analysis. Beam diffraction occurs when a periodic optical structure is formed on/in a substrate. For example, a stripe of light-absorbing zones functions as a diffractive optical element. Therefore, it is theoretically possible to construct a diffractive optical element that is sensitive to chemicals, if a periodic optical structure, e.g., a stripe of color pattern, appears as a result of the response to the chemicals. This sensor would be capable of acting as a transmission grating, and light absorption could then be measured quantitatively by recording the intensity of the diffracted beam. In this case, the signal, i.e., the intensity of the diffracted beam, arises from zero background levels. Such a method, therefore, although based on absorption spectrometry, may be similar in nature to fluorescence spectrometry. In this study, we derive an equation to calculate the intensity of the diffracted beam using a simple model and discuss the merits of this method. To demonstrate the validity of the approach, we constructed a prototype of a diffractive optical chemical sensor that is sensitive to changes in solution pH. THEORY Sensitivity. The model used for calculation of the diffracted beam intensity is shown in Figure 1. The sensor consists of two parts; one is transparent, while the other partially absorbs light. When absorption is negligible, a transmitted beam travels as it does. Then, no diffraction occurs. On the other hand, when strong absorption occurs, a transmitted beam is diffracted as it would pass through a transmission grating. This suggests that the beam diffraction occurs by imbalance of the intensities for the beams (4) St. John, P. M.; Davis, R.; Cady, N.; Czajka, J.; Batt, C A.; Craighead, H. G. Anal. Chem. 1998, 70, 1108-1111. (5) Burggraf, N.; Krattiger, B.; de Mello, A. J.; de Rooij, N. F.; Manz, A. Analyst 1998, 123, 1443-1447. 10.1021/ac990216n CCC: $18.00
© 1999 American Chemical Society Published on Web 05/14/1999
Figure 1. Model for the calculation of the intensity of the beam diffracted by a stripe of light-absorbing zone.
passing through these two parts. Thus, the intensity of the diffracted beam, D, is given by
( )
1 1 1 I D ) ∆I ) I0 - I ) I0 1 2 2 2 I0
(1)
where ∆I is the difference in the intensities for the beams transmitted from the transparent and light-absorbing parts, respectively, and I0 and I are the light intensities before and after beam transmission, respectively. The intensity of the transmitted beam is calculated by Lambert-Beer’s law,
A ) log
(
) ()
(1/2)I0 (1/2)I
) log
I0 I
(2)
where A is the absorbance and is proportional to the product of the molar absorptivity, , the thickness, b, and the concentration, c, of the sample. By substituting eq 2 into eq 1, the intensity of the diffracted beam is given by
D ) (1/2)I0(1 - 10-A)
(3)
When the absorbance is small (i.e., A < 0.02),
D ) (2.30/2)I0A
(4)
On the other hand, the signal intensity in fluorometry is given by
F ) 2.30I0Akφf
(5)
where I0 is the intensity of the light source, φf the fluorescence quantum yield, and k the fluorescence collection efficiency (the detection efficiency is assumed to be unity). Thus, the ratio of the light intensities for the diffracted beam and the fluorescence can be calculated by
fluorescence is collected by an integrating sphere), these methods have the same sensitivity for a sample with a fluorescence quantum yield of 0.5. Typically, the fluorescence quantum yields are much less than 0.5. Moreover, it is difficult to collect all of the fluorescence emitted from a sample. In fact, the efficiency of light collection in fluorometry is in the order of 0.1-0.001 in most cases. However, the present approach permits the collection of nearly all of the diffracted beam. Thus, the present approach, which is based on beam diffraction, can, in practice, be more sensitive than fluorometry. It should be noted that the majority of analytical samples show only slight fluorescence or none at all. Thus, an approach that involves the detection of a diffracted beam is more universal. Several approaches for sensitive absorption spectrometry using lasers have been reported to date. Some of these measure the beam diffracted from the sample. For example, thermal diffraction (grating) spectrometry is used for measurement of small amounts of light absorption.6 However, the signal intensity is proportional to the square of the sample concentration, as well as to the cubic of the laser intensity, and thus is difficult to apply to samples at low concentrations in trace analysis. This situation is identical to other methods, such as degenerate four-wave mixing spectrometry.7,8 The merits of the present approach are as follows: (1) a low-power laser or even a conventional light source such as a xenon arc lamp can be used for trace analysis; (2) the absorption spectrum can be directly measured by means of a white light source and by recording the diffracted beam pattern by an image sensor, to be described in the following section. The disadvantage of the method is straightforward. When the refractive indexes for the transparent and light-absorbing zones are different from one another, a background signal appears. As a result of this, their refractive indexes should be precisely matched. At the same time, the light scattered at zone boundaries should be minimized to reduce the background. Otherwise, the variation of the background signal which occurs as a result of the instability of the light intensity will become a limiting factor for the sensitivity, although such a background signal can partially be canceled using a dual-beam scheme, as in the case of conventional absorption spectrometry. It is also noted that the signal intensity is proportional to the intensity of the light source and a laser with a large output power is desirable. However, a linear detectability will be lost by the refractive index change arising from the heat induced by high-power laser irradiation. Other Parameters. The beam diffraction occurs as a result of the formation of a light-absorbing transmission grating. Then, it is possible to calculate other optical parameters, according to the theory well-known in the optics. For example, the diffraction angle is given by
sin θ - sin θ0 ) mλ/d
(7)
(6)
where θ and θ0 are the angles for the incidence and diffracted beams, respectively, m is the order of diffraction, λ is the
Assuming the most favorable cases for both the approaches (many diffracted beams appear, and except for the nondiffracted beam, all are collected by a concave mirror with a hole; all the
(6) Zhu, X. R.; McGraw, D. J.; Harris, J. M. Anal. Chem. 1992, 64, 710A718A. (7) Wu, Z.; Tong, W. G. Anal. Chem. 1993, 65, 112-117. (8) Nunes, J. A.; Tong, W. G. Anal. Chem. 1993, 65, 2990-2994.
D/F ) 1/2kφf
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Figure 3. Beam diffraction patters. Condition: (a) bare sensor; (b) sensor dipped in distilled water; (c) sensor dipped in alkaline solution. The spot size of the laser at the diffractive optical element is ∼1 mm. Thus the number of elements in the laser irradiation area was 2. The optical density of the light-absorbing zone was not measured, but it was visually confirmed to be in the order of A ) 1.
Figure 2. Protocol for the construction of diffractive optical chemical sensor sensitive to pH change.
wavelength of the light, and d is the spacing of the diffractive optical element. This equation suggests that the diffraction angle of the first-order beam is θ ) 0.0725° when a He-Ne laser (λ ) 633 nm) is introduced perpendicularly (θ0 ) 0) to a diffractive optical element with a spacing of d ) 500 µm. The resolution (R) is given by a product of the diffraction order (m) and the number of lines (N) in the laser illumination area (W). For example, the resolution is 2 for the diffractive optical element used in this study, i.e., R ) mN ) m(W/d) ) 1 × (1 mm/0.5 mm) ) 2. It is noted that, even for a value of R ) 1 (a rectangular hole), an interference pattern is observable and can be used for the detection of light absorption. When this absorption detector is applied e.g., to capillary electrophoresis, in which the channel width is ∼50 µm, the resolution is 20. The diffraction angle depends on the wavelength of the light introduced, and as a result, the sample itself acts as a diffraction grating in conventional absorption spectrometry. The resolution (R ) 20) in the above example (e.g., ∆λ ) 25 nm at λ ) 500 nm) is not very high but is still acceptable for recording the absorption spectrum of the sample in the condensed phase. Needless to say, it is possible to increase the resolution by expanding the laser illumination area and/or reducing the channel width. The maximum resolution is limited to the minimum spacing of the diffractive optical element, as in the case of a conventional grating. As calculated from eq 7, d ) λ for m ) 1 and θ ) 90°. This indicates that the minimum spacing is determined by the wavelength of the light introduced. 2264 Analytical Chemistry, Vol. 71, No. 13, July 1, 1999
EXPERIMENTAL SECTION pH Sensor. Figure 2 shows the protocol for construction of a prototype pH sensor based on beam diffraction. First, a metal template consisting of a grid spaced by 0.25 mm is placed on an ordinary microscope slide. A gelatin solution (7%) containing the pH indicator, thymolphthalein, which is transparent at pH 10.5, was dropped onto the template and covered with another microscope slide. This was placed in the refrigerator until the gelatin became a soft gel. The upper microscope slide and the template were then removed, and the gel, which remained on the bottom microscope slide was used as a pH sensor (sensor 1). Second, a gelatin solution (7%) containing no pH indicator was dropped onto sensor 1 to fill the vacancies between the grooves of the gelatin. This was covered with another microscope slide. A thin polyethylene film was inserted between the gelatin layer and the upper microscope slide, to avoid their adhesion, and the assembled materials were placed in the refrigerator again until the gelatin became a soft gel. The upper microscope slide and the polyethylene film were removed, and the gel that remained was used as a pH sensor (sensor 2) without removing it from the bottom microscope slide. Analytical Instrument and Procedures. The constructed pH sensor was immersed in distilled water in a quartz cell. The solution, which was acidic due to the nature of the gelatin, was then made alkaline by adding several drops of a NaOH solution. A He-Ne laser (Uniphase, 2 mW) emitting at 633 nm was used as a light source. The intensity of the diffracted beam was measured by a photodiode equipped with a digital oscilloscope (Yokogawa, DL1200E). The diffraction pattern was recorded by a digital camera (Fuji, DS8) and was processed on a personal computer. RESULTS AND DISCUSSION When the He-Ne laser was passed through sensor 1, dipped in distilled water, an intense pattern consisting of numerous spots
arising from diffracted beams were observed. By changing the solution from acidic to alkaline, a blue stripe appeared and the intensity of the diffracted beam increased ∼10%. The strong background signal arises from a difference in the refractive index for the parts with and without the gel. To reduce this background signal, the vacancies between the gelatin grooves were filled with a gelatin that contained no indicator. The pattern of the beam transmitted from this sensor (sensor 2) in air is shown in Figure 3a. The strong spot observed at the center indicates that the amount of diffraction is very small or absent, since the effective optical path lengths are nearly identical to each other for the parts with and without the indicator reagent. When the sensor is dipped into distilled water, no appreciable change is observed in the beam pattern as shown in Figure 3b, since thymolphthalein is colorless in acidic solution. However, when the solution was made alkaline, a blue stripe appeared since thymolphthalein becomes blue at these conditions. The beam pattern observed is shown in Figure 3c. Many spots arising from the diffraction of the He-Ne laser beam appear. Thus, pH change can be detected by observing the generated diffraction pattern. The present approach has unique capabilities. It allows for the display of the analytical result without any detector, electronics, or recorder. All the measurements are performed optically, and no electricity is required except for the generation of the laser. The present situation is, of course, similar to visual conventional
colorimetry. This approach, however, permits an on-site display of the analytical results using letters and figures. For example, it is possible, in theory, to transmit the laser beam through a long distance from the detection area using an optical fiber and to display the analytical result directly on a large screen or even the wall of a building by projecting letters, e.g., “Alkali! Danger!”. Needless to say, this method could be useful for sample detection at hazardous places, under strong radio frequency interference noise, or even in an atmosphere of explosive gases. Thus the present method may be successfully used in remote sensing in conjunction with on-site display. Many potential applications for this diffractive optical chemical sensing, which is based on light absorption, exist. For example, recently developed capillary electrophoresis requires a versatile absorption detector that is as sensitive as a laser fluorometric detector, especially in the deep ultraviolet region (e.g., 200 nm) where no suitable continuous-wave laser is available. Thus, the present detection scheme may be advantageously used in such applications, which require high sensitivity in practical trace analysis.
Received for review February 23, 1999. Accepted April 20, 1999. AC990216N
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