Characteristics of photon detectors in reference [17] and [18]
1. Introduction
In biological research, the luminescience from fluorescent proteins or luminescent enzymes is widely applied for monitoring a change of environment at a cell. Biomolecules used as the probe, such as Green Fluorescence Protein (GFP) or luciferase molecules found in fireflies can respond to the existence of specific molecules or ions and subsequently emit a photon. The detection of a specific molecule can then be confirmed by detecting the emitted photons efficiently with a photon detector. A highly efficient detection of the luminescence is normally essential to realization of a high sensitivity to the specific molecules or ions and an improvement of the sensitivity can upgrade the capability of detection in a low concentration of sample solution. Therefore, there are many efforts to improve the efficiency of the collection of emitted photons and of the optical coupling to the photon detector.
A straightforward method is to directly detect the luminescence from the sample solution in a test tube with a single photon detector via simple coupling optics as shown in Fig. 1. This detection system is very simple and easy-operational, so that it has been widely used for various applications so far. To realize high efficiency detection, however, this method needs a single photon detector with the wide photon-sensitive area, which is ideally larger than a photon-emission area in the test tube. Here, we are introducing an alternative method, where the luminescent biomolecules are immobilized at an optical fiber end and the luminescence is detected by a photon detector which is optically coupled to the other optical fiber end. Fig. 2 illustrates the optical fiber-based system. This method has been investigated for application to a fiberoptic biosensor, which is constructed by immobilizing either an enzyme or an antibody. A review of this method is given in reference [1] and [2].
This method has three merits. The first one is to permit a local detection within the sample solution, because the optical fiber end functions as a needle-like probe. Meanwhile, the method as shown in Fig. 1 is suitable for detecting the luminescence from a large area in the sample solution. The second one is that the detection scheme does not require that the photon detection is very close to the sample solution. This feature makes it easier to mount the sensing parts in integrated bioengineering, such as
We have built detection systems of bioluminescence at an optical fiber end and investigated the sensitivity of Adenosine triphosphate (ATP) detections by using an APD-type as described in [3] and [4]. In this chapter, results with a PMT-type detector are presented in comparison with the results by using the APD-type photon detector. We also discuss the reason of limiting the present sensitivity in the system with the PMT-type detector. ATP is a good indicator of biochemical reaction or life activity, since ATP is considered as the universal currency of biological energy for all living things. Therefore, there are many efforts to develop ATP-sensing techniques for compact and efficient ATP detection in reference [5-7]. In particular, high-sensitivity detection of ATP can indicate the existence of microorganisms even in low numbers. Thus, a compact, simple, and easy-operational system with extremely high sensitivity has been desirable.
One well-known and powerful method for highly sensitive ATP detection is to use the chemical reaction involved in thebioluminescence, the luciferin-luciferase reaction in reference [8]. In this reaction, after one ATP molecule and one luciferin molecule are bound to one luciferase molecule, and the luciferin molecule is oxidized using the energy of ATP. As consequence, one photon is emitted during the transition from the excited state to the ground state of the oxidized luciferin molecule bound to the luciferase molecule. The emission of one photon indicates the use of the energy of one ATP molecule. In the method using the luciferin-luciferase reaction, the efficient detection of the bioluminescence is essential for high-sensitivity detection of ATP.
The oxidation of luciferin is catalysed by the enzyme luciferase, so that the immobilization of luciferase molecules on solid probes of various sizes allows highly sensitive and local detection of ATP. Three types of immobilization have been used: firstly attachment to the cell surface in [9], secondary attachment to small particles, such as nanoparticles in [10], glass beads and rods in [11], thirdly attachment to extended objects with a size in the centimeter range, such as strips in [12] and [13], and films in [14]. For the ATP-detection on the intermediate scale below 1 millimeter, a fiberoptic probe employing immobilized luciferase in [2] as well as microchips in [15] and [16] is utilizable. Therefore, the efficient detection system of bioluminescence at an optical fiber end can achieve the local detection of ATP. The realization of highly sensitive detection of ATP potentically provides the local detection of extremely low number of microorganisms. Thus, it is desirable to construct a highly efficient detection system of the bioluminescence at an optical fiber end and to evaluate the detection limit with the system. In order to explore possibilities for improving the detection limit, moreover, it is also necessary to investigate the bioluminescent reaction at an optical fiber end.
The rest of this chapter is organized as follows. In sec. 2, we describe a concept for the construction of the optical fiber-based system and show how to construct the detection systems by using the PMT detector. In sec. 3, we describe the sensitivity test with the constructed system and show the results are consisitent with the APD, but also show that the sensitivity can not reach the expected detection limit. In sec. 4, we present the results of kinetic properties obtained from experimental data on the bioluminescence and clarify a dominant reason of restricting the detection limit.Sec. 5 summarizes prensent results and future problems.
2. Construction of the optical fiber-based system
2.1. General concept
For the construction of an efficient detection of a bioluminescence, it is necessay to consider a collection efficiency of the luminescence at the optical fiber end and a coupling efficiency between the other optical fiber end and a photon detector as described in [4]. Using the optical fiber with a core diameter
From the simple calculation of the solid angle with a maximum open angle
where
In the following, let us consider the situation where the other optical fiber end is optically coupled to a photon detector with a detection window of diameter
It should be noted that the coupling efficiency
2.2. Construction with a cooled PMT detector
2.2.1. Photon detectors
To construct the optical fiber-based system, a choice of a single photon detector is very important. Photon detectors generally have two significant factors contributing to the sensitivity of detection for weak light: the efficiency and the dark counts of the detector. Recently, two types of single photon detectors, which are a cooled APD and a small size of cooled PMT, are available. The cooled APD which can detect for single photons is mostly used because of the high quantum efficiency and the low dark counts. The sensitive area must be very small (
We selected the PMT counting head (H7421) manufactured by Hamamatsu Photonics K.K. for this system. Its characteristics are summarized in Tab. 1. For comparison, we also present the characteristics of the APD-type photon counting module (SPCM-AQR-14) provided by Perkin Elmer. Ltd. It has already been verified that this APD is applicable to the fiber-based system by our previous investigation as discussed in [3], [4].
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PMT (H7421) | 40% at 550nm | 100 s-1 | 5.0 mm | 0.123 | 7.2 mm |
APD (SPCM-AQR-14) | 55% at 550nm | 100 s-1 | 0.175 mm | 0.78 | 6.16 mm |
The value of
2.2.2. Coupling efficiency
As showing in section 2.1, under the condition of
The coupling efficiency
On the other hands, in the use of the APD, the optimization of
The relative sensitivity for the bioluminescence detection can be compared by using a product of FOM and the quantum efficiency
3. Sensitivity test for ATP detection
3.1. Luciferin-luciferase reaction
Bioluminescence in living organisms, such as fireflies and some marine bacteria, typically occurs due to the optical transition from the excited state to the ground state of oxidized luciferin molecules produced by the luciferin-luciferase reaction under the catalytic activity of luciferase molecules. This reaction can be expressed by the following squential of reaction steps:
where E indicates luciferase,
where
In the presence of enough luciferin molecules, the immobilization of luciferase molecules at the optical fiber end allows us to sense the present of ATP around the fiber end using single photon counts. For this purpose, we used a compound protein containing a silica-binding protein ( SBP ) molecule and a luciferase molecule ( SBP-luciferase ), which were recently synthesized by Taniguchi and co-workers in [23]. This protein makes it possible to immobilize a lucuferase molecule on the optical fiber end through a SBP molecule retaining its activity. The spectrum of the emitted photons shows a central wavelength of 550 nm and a width of about 100 nm in reference [24] and [25]. Both photon detectors of the APD and the PMT have the large quantum efficiency at 550 nm, the photon counting detectors are suitable for ATP sensing.
3.2. Michaelis-Menten formula
In the solution containing nonlocalized homogeneously dispersed luciferase and ATP, the Michaelis-Menten formula is applicable to the enzyme reaction as descibed in [26]. In the presence of sufficient luciferin molecules in the solution, a rate of emitted photons at steady state
where
3.3. Mesurement of the sensitivity
3.3.1. Experimental setup
Fig.9 shows the experimental setup for the sensitvity test and the investigation of bioluminescence at the optical fiber end. One optical fiber end was optically connected to the PMT as describing in the previous section. On the other fiber end, the luciferase molecules were immobilized via SBP molecules and the bioluminescent reaction occurs by immersing the luciferase-terminated fiber end into a sample solution. The emitted photons were transmitted to the PMT through the optical fiber and TTL pulses outputted from the PMT were counted by a PC card installed in a personal computer(PC). To reducing the background light, the whole system was put into the dark box.
For the observation of the bioluminescence rising, a test tube containing the sample solution was fixed on the Z-stage, which is a motorized stage and externally controllable. By raising the test tube for immersing the luciferase-terminated fiber end after starting the data acquisition system, the photon counts rising from the background level and subsequently reaching a maximum were observed with time. The numbers of detected photons during 0.0298 s or during 0.1 s were recorded every 0.0321s or every 0.1s by the PC, respectively. These values were obtained from a calibration test of data acquisition system. The details on the experimental setup and the measurements are described in reference [28].
3.3.2. Immobilization of luciferase
Before immobilizing luciferase molecules, we cut the optical fiber and cleaned the cut surface with ethanol and Tris buffer (0.25mM Tris-HCl with 0.15 M NaCl). Different from the previous experiments with the APD, we cleaved the optical fiber for making a flat surface on the fiber end, which can reproduce the number of immobilized luciferase. The flat surface also allows us to indivisually evaluate the number of immobilized luciferase molecules by using element analysis, although the sensitivity with the flat surface is about 10 times lower than the one with the appropriately irregular surface cut without the cleaving technique as described in [29]. The cut surface was direcly observed by using the fiber scope and checked its flattness by eyes. After cleaning, the surface was immersed in a solultion of SBP-luciferase and was left at a temperature of
For evaluating the number of immobilized luciferase molecules, element analysis to the fiber end was carried out by using Xray Photoemission Spectroscopy (XPS). We measured a spectrum including peaks from nitrogen in the SBP-luciferase and from silicon on the surface of the fiber end which made with the silica and obtained the ratio of the area of the nitrogen-peak to the one of the silicon-peak. Utilizing the absolute number of silicon on the surface of the fiber end, the surface density of immobilized luciferase molecules was determined to be
3.3.3. Sample solutions
The samples were a 1:4:4:31 mixture of 20 mM D-luciferin solution, Tris buffer solution( 250 mM Tris-HCl mixed with 50 mM MgCl2 ), ATP solution, and distilled water. Several solutions of ATP with different ATP concentrations were made by diluting the ATP standard in ATP Bioluminescence Assay Kit CLS II manufactured by Roche Co. Ltd. A series of sample solutions with different ATP concentrations were prepared in advance. To obtain a background before the ATP measurements, an additional sample without ATP was also produced by mixing distilled water instead of the ATP solution.
3.3.4. Results
The time dependence of photon counts per 0.1-s interval were measured in immersing the luciferase-terminated fiber end into the sample solutions with various ATP concentration and converted to the values of photon counting rate. A typical result for
The photon counts rise up, reach a maximum at about 100 s, and decrease toward the background level with time scale of 1000 s after the immersion. The background level was about
Statistical errors were estimated as one standard deviation assuming Poisson distribution. From Fig. 11, the sensitivity in this system is limited to
To check the ATP concentration dependence of the photon counting rate at maximum, the average of counts in sixteen 1-s intervals around the time at which the counting rate become maximal was calculated for each ATP concentration. The results are indicated by the solid circles in Fig. 12. By the analysis of fitting data points in Fig. 12 to Eq. (3), we obtained the Michaelis constant of
3.3.5. Discussion
The detection limitis essentially determined by both of the parameter
In the PMT system, it is noted that the improvement of two orders of magnitude for
4. Investigation of bioluminescence at the optical fiber end
4.1. Measurement of the bioluminescence with high time resolution
To obtain the reaction rate
Fig. 13 (a) shows the result of the detected photons with the immobilized SBP-luciferase molecules at the optical fiber end into the solution of
4.2. Analysis
4.2.1. Reaction model including inhibitors
For obtaining kinetic parameters of bioluminescient reaction, we consider the rate equations including the effects of inhibitors. In the luciferin-luciferase reaction, two kinds of products, oxyluciferin and
Here,
In the use of the immobilized luciferase molecules for sensing dispersed ATP molecules, it is natural to consider that the reaction occures in a volume
where the variable
In addition to the above rate equations, the following conditions described as,
should be satisfied. The condition of Eq. (11) shows that the total number of active luciferase molecules
Using Eq. (11), Eq. (12), Eq. (13), and Eq. (14) as boundary conditions and inputting the constant values of
In the solution containing non-localized homogenous dispersed SBP-luciferase, the volume of
4.2.2. Results of analysis
The five parameters
The chi-square
The result of fitting the data from 0 s to 30 s is represented as a solid line in Fig. 13 (a), which is extrapolated to 60 s using the obtaind parameters. This result is not reproduced completely in the time range from 0 s to 60 s, because the effect of the competitive inhibition of oxyluciferin is not considered and the fitting fuction includes only the contribution of the deactivation process. The inhibition of the oxyluciferin weakens with time due to its diffusion process, but this diffusion effect is not considered in this analysis. In contrast, the contribution of the deactivation process, which was evaluated by fitting the data around the peak, is concequently overestimated compared to the actual contribution. Therefore, the effect of the relatively strong evaluation for the deactivation process appears in the time range after 30 s.
The parameters obtained by fitting the data are summarized in table 2 together with the results of the dispersed luciferase for comparison. The parameter
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0.44 | 0.010 |
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4.3. Discussion
In table 2, it is easily seen that the kinetic parameters in the immobilized luciferase are almost same as in the non-localized dispersed luciferase except the reaction rate of
On the reaction rates, the obtained value of
From table 2, the activation ratio
5. Summary
We introduced a method of high-sensitivity detection of bioluminescence at an optical fiber end for an ATP detection as an efficient alternative to direct detection of bioluminescence for a sample solution. For investigation of the bioluminescence, we constructed an optical fiber-based system, where the luciferase molecules are immobilized on the optical fiber end and the other end is optically coupled to a compact size of cooled PMT-type photon counting head which has a large sensitive area. Although the sensitivity for the bioluminescence is not optimal, it is almost same as the system which had been constructed with an APD-type photon counting detector. We have evaluated the sensitivity for ATP detection and verified the detection limit of
Acknowledgements
We are grateful to Prof. Hiroyuki Sakaue for supporting the element analysis with XPS and Prof. Kenichi Noda for useful supports to the experiments. This work has been partially supported by the International Project Center for Integration Research on Quantum, Information, and Life Science of Hiroshima University and the Grant-in-Aid for Scientific Research (C)(19560046) of Japanese Society for the Promotion of Science, JSPS.
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