Single-Beam Optical Biosensing Based on Enzyme-Linked Laser

Oct 22, 2012 - Fresnel equation qualitatively reproduce the experimental results, indicating that the backreflected laser oscillation occurs because o...
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Single-Beam Optical Biosensing Based on Enzyme-Linked Laser Nanopolymerization of o‑Phenylenediamine Hiroyuki Yoshikawa,* Shuhei Imura, and Eiichi Tamiya Department of Applied Physics, Osaka University, Suita, Osaka 565-0871, Japan S Supporting Information *

ABSTRACT: We report an innovative biosensing technique using a focused laser beam for the fabrication of a polymer nanostructure and the detection of its nanoscale growth. A nanoaggregate structure is formed by focusing a single beam of a continuous-wave (cw) green laser beam on an o-phenylenediamine (o-pD) solution dropped on a glass substrate. The backreflection intensity of the focused laser beam shows a temporal oscillation, whereas the size of the aggregate monotonously increases. Simple calculations based on the Fresnel equation qualitatively reproduce the experimental results, indicating that the backreflected laser oscillation occurs because of the interference between two beams reflected at the front and the back surfaces of the aggregate. Because the growth speed of the aggregate depends on light absorption by the oxidized o-PD, the backreflection oscillation curve can be used to monitor the oxidative reaction in the solution. We apply this phenomena to optical biosensing based on the oxidation of o-PD by the peroxidase enzyme reaction. A reliable quantification of glucose can be achieved by simply focusing a single laser beam and detecting its reflection intensity in a manner similar to the optical pickup unit of optical storage drives. anotechnology has contributed greatly in the field of biosensing research.1−9 Nanostructures not only play an important role in the transduction of biomolecular interactions into detectable and quantifiable signals but also have considerable merit in highly sensitive, high-throughput, rapid, and ultramicro analysis. Many researchers in this field spend a great deal of time, effort, and money to make up nanostructures using one or more nanofabrication techniques including electron beam lithography,7 plasma processing,8 and nanoimprinting.9 In addition, specialized and expensive instruments are indispensable for highly sensitive detection. Thus the trend of biosensing research is going to require technical skills, expensive instruments, or time-consuming and complex procedures more and more. This current situation is contrary to an urgent need for the development of biosensors that can be operated even by untrained personnel for applications in point-of-care diagnostics, food safety, and environment monitoring. We introduce here a novel nanobiosensing technology using a single laser beam and enzyme reactions. In 1962, Clark and Lyons10 first proposed the amperometric detection of glucose by use of a glucose oxidase (GOD) immobilized electrode. In the years since, various biosensors based on enzyme reactions have been studied and developed. In currently available biosensors, the signals obtained by enzyme reactions are mainly transduced into electronic or optical signals.11−16 In the latter case, enzyme reactions produce or change the absorption or fluorescence of color reagents. One drawback of such optical biosensors is that their size and cost can be reduced to only a limited extent, owing to the fact that the detection of fluorescence or absorption

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© 2012 American Chemical Society

changes necessarily requires optical spectroscopic instruments such as a spectrometer, multichannel detector, or optical filters. Nowadays, there is an urgent need for the development of portable biosensors that can be operated even by untrained personnel as mentioned above. In this study, instead of detecting enzyme reactions by monitoring the absorption or fluorescence, as is typically done, we detect them by monitoring the backreflected light of a focused laser beam. The key is the oxidative polymerization of o-phenylenediamine (o-PD), which is commonly used to transduce enzyme reactions into a color change, that is, an absorption or fluorescence spectral change, in conventional methods. We noticed that this oxidative polymerization is detectable as a reflection or scattering intensity of light by making the size of polymerization products comparable to the wavelength of light. The focused laser beam promotes the oxidative polymerization of o-PD molecules, leading to the formation of a polymer nanoaggregate whose size is precisely reflected by the intensity of the backreflected light. Optical biosensors based on peroxidase enzyme reactions can be constructed by simply focusing a single laser beam and detecting the backreflection intensity. This detection scheme is simple and yet enables rapid and high-sensitivity microanalysis. Received: July 11, 2012 Accepted: October 22, 2012 Published: October 22, 2012 9811

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Figure 1. (a) Schematic of the experimental setup. (b) Bright-field image of the spot formed on the glass substrate after focusing the laser beam. (c, d) Scanning electron micrographs of the spot: (c) top view and (d) 45° view.

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EXPERIMENTAL SECTION o-PD was purchased from Sigma and used without further purification. o-PD stock solution (1 mM) was prepared by dissolving o-PD in pure water and storing in a refrigerator. For glucose detection, citrate buffer (pH 4.6) was used as a solvent. Horseradish peroxidase (HRP) (Wako, >100 units/mg) and glucose oxidase (GOD) (Toyobo Enzyme, >100 units/mg) were dissolved in citrate buffer (pH 4.6) at 0.01 and 0.1 mg/ mL, respectively. HRP and GOD stock solutions were mixed in the ratio 1:1 immediately before use. The absorption spectra of sample solutions were measured by using a UV−visible absorption spectrometer (Shimadzu, UV-2550). The topography and morphology of the produced nanostructures were evaluated by using an atomic force microscope (AFM) (SII, SPI-4000) and a scanning electron microscope (SEM) (FEI, DB-235). Figure 1 shows a schematic of the experimental setup. A green (wavelength 532 nm) diode-pumped solid-state (DPSS) laser beam (Shanghai Dream Lasers, SDL-532-020TL) was made incident on an optical inverted microscope (Olympus, IX70) via a beam expander. The laser beam was focused by using an objective lens (60×, N.A. 0.9). The x−y position of the sample and the laser irradiation time were controlled by a motorized stage and a mechanical shutter, respectively. The Rayleigh scattering spectrum of a single aggregate was measured by using a multichannel spectrometer (B&W Tek i-trometer) under the dark-field illumination of a halogen lamp. The backreflected light intensity of the laser beam was detected by using a photomultiplier (Hamamatsu Photonics, R1166).

shows scanning electron microscope (SEM) images of this spot. Interestingly, it was seen that the aggregates comprised smaller nanoparticles. The surface of the aggregate was covered by the nanoparticles, but the outline had a regular domelike shape. The radial size of the aggregates corresponds to that of the laser focus (∼800 nm), indicating that the chemical reaction is significantly accelerated only at the laser-irradiated spot. If this reaction were to be attributed to the thermal effect, aggregates much larger than the laser spot would be formed because of the thermal conductivity of the solvent and the glass substrate. Therefore, it is likely that the chemical reaction, that is, photochemical oxidative polymerization, is induced by the focused laser beam. Because aniline derivatives have no absorption in the visible wavelength region, previously photocatalytic compounds such as ruthenium complex and methyl viologen were used to initiate their photopolymerization.17,18 In the present study, no photocatalytic compound is added to the o-PD solution, that is, there are no apparent absorptive species that initiate the photochemical reaction. However, natural oxidation is unavoidable under atmospheric conditions. Because a small amount of o-PD can be oxidized by atmospheric oxygen dissolved in water, the o-PD solution has a weak absorption band at ∼440 nm, as shown in Figure S-1 (Supporting Information). This band is attributed to o-PD dimers (2,3-diaminophenazine).17,19,20 In the present case, it is conceivable that o-PD dimers absorb the laser light and initiate the reaction. Because it is impossible to measure the absorption spectrum of a single nanomaterial, the spectrum of the scattered light of the aggregate deposited by the focused laser was measured under dark-field illumination. As shown in Figure S-1 (Supporting Information), the peak of the scattering spectrum is located at ∼620 nm, which is much longer than the absorption peak of o-PD dimers in the solution. This means that the aggregate is composed of polymerized molecules whose π-conjugation length is longer than that of the dimers. Further investigation is necessary to explore the reaction mechanism in detail. Next, we investigated the characteristic behavior of the backreflected light during this reaction process.

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RESULTS AND DISCUSSION o-PD is a reagent that forms a colored product upon oxidative polymerization.12 First, we investigate the effect of focusing a laser beam on an o-PD aqueous solution. The solution was dropped on a microscope cover glass, and a 2-mW green laser beam was focused on the glass−solution interface, as shown in Figure 1a. Figure 1b shows an optical microscope image of a small spot formed at the laser focus position after laser irradiation of the 1 mM o-PD solution for 80 s. Figure 1c,d 9812

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Figure 2. (a−f) Sequence photographs of the backreflected laser beam taken every 4 s in the 1 mM o-PD solution. (g) Temporal variations of the backreflected laser intensity at different o-PD concentrations (black, 0.5 mM; red, 1 mM; blue, 4 mM). The detection area is ∼3 μm around the laser focus.

Figure 3. (a) An AFM image of poly(o-PD) nanoaggregates deposited at different laser irradiation times (4, 6, 8, 10, 12, 14, 16, 18, and 20 s, from the upper right corner to the lower left corner). The concentration of the original o-PD solution is 4 mM. (b−d) Enlarged AFM images of nanoaggregates deposited by laser irradiation for 4, 10, and 16 s. (e) Height of each aggregate and temporal variation of the backreflection, plotted vs laser irradiation time.

Figure 2a shows the backreflected laser spot monitored by a charge-coupled device (CCD) camera attached on the microscope. The intensity of the green laser spot on the

monitor gradually increased after laser irradiation. However, we noticed that the intensity decreased after some time. This temporal variation of the intensity was recorded by using a 9813

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Figure 4. Polymer thickness dependence of the reflected light intensity from a model polymer film on a glass substrate. The medium on the film is water (n = 1.33). The 532-nm light normally incident from the glass side and the reflectance of the backreflection (180°) were calculated from Fresnel’s equation. The (a) real part and (b) imaginary part of the polymer refractive index (n − ik) were varied.

program. The complex refractive index of poly(o-PD) is varied as a parameter to fit the experimental results. Figure 4 shows that the backreflection intensity versus the poly(o-PD) thickness exhibits a damped oscillation curve. The calculated curves qualitatively correspond to the experimental result shown in Figure 3e. Although there was no report on the refractive index of the poly(o-PD) film as far as we know, optical properties of a polymer film of a similar aniline derivative, o-aminophenol, were studied by ellipsometry in the previous work.22 The complex refractive index of 1.699−0.175i reported in the literature shows good correspondence with that used in our calculations. This indicates the validity of our calculation analysis. In the actual experiment, however, the refractive index may increase with the growth of the aggregate because of the increase in the degree of polymerization. The curvature of the wavefront of the backreflected light is also not considered in this calculation. Although it is difficult to reproduce the experimental curve completely, this calculation provides valuable information. Figure 4 shows that with an increase in the real or imaginary part of the refractive index, the intensity of the peak height increases. Such a difference in the peak height is actually observed in some experimental data, as shown in Figure 2g. A higher o-PD concentration gives a higher rhythm and a higher peak of oscillation, demonstrating that the growth speed and the refractive index of the aggregate increase with the o-PD concentration. It is noteworthy that the nanoscale growth of the aggregate can be measured in real time based on the optical interference by such a simple approach. This is the essential point to realize the following single-beam biosensing based on enzyme reactions. The enzymatic oxidative polymerization of aniline derivatives is a well-known reaction.23 In particular, horseradish peroxidase (HRP) is used to catalyze the oxidative polymerization of o-PD with hydrogen peroxide.19 By combining the generation of hydrogen peroxide with the GOD reaction, the glucose

photomultiplier. Interestingly, it exhibits a smooth oscillation curve, as shown in Figure 2g. Curves obtained from 4 mM (blue) and 1 mM (red) solutions show second peaks that have smaller heights than the first ones. However, the backreflected intensity gradually increases after the second peak. This increase with extended laser irradiation is attributed to the light scattered by the generation of a large roughness or irregularly shaped aggregates, as confirmed by SEM images taken after extended (80 s) laser irradiation for a 4 mM o-PD solution (Figure S-2, Supporting Information). Apart from the light scattering shown in extended laser irradiation, the temporal variation curves of the backreflection showed clear oscillations. To investigate the reason for this oscillation, aggregates deposited with different laser irradiation times were measured by atomic force microscopy (AFM). Figure 3 shows that the height of the aggregate monotonously increases with the laser irradiation time, whereas the backreflection intensity fluctuates. Figure 3e shows that the backreflection intensity reaches a maximum when the height of the aggregate is ∼80 nm and a minimum at a height of ∼180 nm. The relation between the backreflection intensity and the height of the aggregate suggests the following mechanism. The regular backreflection of normal incident light is attributed to a product of two beams that are reflected at the front and back sides of the aggregate. As the aggregate grows, the phase difference between these two beams changes. Thus, the intensity of the observed backreflected light is maximum when the two phases match and minimum when they do not match (half-wave difference). We performed a simple calculation based on the Fresnel equation to verify this explanation.21 In the present case, the backreflected laser intensity is approximated by the light reflection from the glass− poly(o-PD)−water structure as a first approximation. Figure 4 shows the poly(o-PD) thickness dependence of the reflectance as calculated from the Fresnel equations by use of a Fortran 9814

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Scheme 1. Reaction Scheme of Glucose with GOD, HRP, and o-PD

Figure 5. Demonstration of enzymatic glucose sensing with (a, b) backreflected light detection and (c, d) conventional absorption measurement. (a) Temporal variations of the backreflected light at different glucose concentrations. (b) Peak times of temporal variation curves vs glucose concentrations. (c) Absorption spectra at different glucose concentrations. (d) Peak absorbance values plotted vs glucose concentrations.

concentration can be determined from the polymerized products of o-PD. This reaction process is described in Scheme 1. o-PD dimers (2,3-diaminophenazine) are the main product of this reaction, and their color (absorbance) is used to determine the substrate concentration in the conventional approach.12 As discussed above, the amount of dimer determines the growth rate of the aggregate, which is reflected in the temporal change of the backreflected laser intensity. Therefore, we attempted to detect the glucose concentration by the backreflection measurement in the presence of GOD, HRP, and o-PD, as follows. In biosensing applications, enzymes and o-PD are dissolved in citrate buffer (pH 4.6) to ensure the reproducibility of the backreflection curve by stabilizing the enzyme reaction and suppressing the progress of the natural oxidation of o-PD. Actually, the backreflection curve of the oPD aqueous solution varied daily even if the solution was kept in a refrigerator in the dark. The degree of natural oxidation is important to keep the reproducible data for a long period. An o-PD solution with an absorbance of ∼0.08 at 1 mM was

prepared by irradiating a 100-mW green light-emitting diode (LED) light on a fresh solution for a few to several hundred seconds. We confirmed that the same reflection curve was obtained over 1 week by using this solution. Twenty microliters of glucose solution was added to the same amount of enzyme solution, which was prepared by dissolving GOD and HRP in the citrate buffer. After incubation for 1 min, 20 μL of 1 mM oPD was mixed in it. Then, 10−20 μL of the mixed solution was dropped on a glass plate, and the laser beam was focused on it. We confirmed that the result is independent of the amount of the sample solution in this range (10−20 μL), because the solution volume is sufficiently large. When the molecular diffusion under the 100 s laser irradiation is taken into consideration, ∼0.4 μL sample solution would be enough to make a nanoaggregate in the laser focus. Four different concentrations of glucose and 5 mM ribose and lactose were used as the target molecules. As shown in Figure 5, the temporal variations of the backreflected laser intensity obviously depended on the glucose concentration. The peak 9815

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time of each curve was plotted as a function of the glucose concentration in Figure 5b. The measurements were repeated three times and the average values were plotted with standard deviations. Interestingly, 5 mM ribose and lactose gave a signal of the same level as 100 nM glucose. The activity toward ribose and lactose is 0.00% of that toward glucose according to the specification sheet of GOD. The experimental result may indicate a weak activity (∼0.002% of the glucose activity) toward ribose and lactose, which is difficult to estimate in the conventional way. This is otherwise attributed to glucose originally included in their reagents as impurities. Although a further investigation is necessary to reveal the origin of this weak nonspecific response, Figure 5b shows that the glucose concentrations are quantitatively determined in the range between 1 mM and 100 nM from the peak times with high specificity. The high sensitivity is supported by the quality of the signal. Thanks to the regular shape of the signal curve, the curve peak could be decided by a linear regression fitting. However, the high concentration glucose shows an irregular shape of the reflection curve without a clear peak, because of the immediate formation of a large and irregular shape aggregate as shown in Figure S-2 (Supporting Information), making it impossible to quantify concentrations higher than 1 mM by peak detection. The higher glucose concentration becomes measurable by reducing enzyme or o-PD concentration, although sensitivity is sacrificed. In the conventional approach, the light absorption given by o-PD dimers (2,3-diaminophenazine) is used to determine the substrate concentration. We compared our method with the conventional one under the same conditions, that is, the same enzyme concentration and incubation time. As shown in Figure 5c, d, it is difficult to distinguish the signals at low glucose concentrations in the conventional absorption measurement. The limit of detection (LOD) of the conventional absorption detection is 100 μM, whereas that of our present method is 100 nM. The sensitivity of our method is quite high, even compared with that of other glucose biosensors reported in literature.11,13,24−26 In addition, the detection is carried out with a shorter response time compared with previous works that used enzymatic o-PD reactions.11,12 A focused laser beam may positively affect the formation of molecular assemblies because of the optical force or local temperature elevation, as reported in previous studies.27−29

compact and inexpensive optical biosensors for widespread applications to point-of-care monitoring.

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ASSOCIATED CONTENT

S Supporting Information *

Additional text and two figures describing spectroscopic evaluation of o-PD solution and the single poly(o-PD) aggregate, backreflection curves, and SEM images obtained at different o-PD concentrations. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by JSPS, KAKENHI (23685016). REFERENCES

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CONCLUSIONS In this study, we found a characteristic modulation of the backreflected laser intensity induced by focusing a laser beam on an o-PD solution, and we applied this to glucose detection. Because enzyme reactions using HRP are widely used for biosensing applications, particularly in enzyme-linked immunosorbent assays (ELISAs), this technique should also be applicable to various biosensors. A reliable optical quantification of glucose can be performed in a short time (∼2 min including incubation) with a small sample volume (