Development of a highly sensitive microplate luminometer using ATP bioluminescence.

Analytical techniques using ATP bioluminescence, which has a high quantum yield and substrate specificity, are widely used in various assays, such as luciferase reporter assays, in the biological sciences. Although most microplate luminometers can be used to measure ATP luminescence with 96-well or 384-well microplates, their ATP detection limits are typically several tens of amol, which is not sufficient for evaluating cell activities and variability within small samples, such as those containing only a few cells. To analyze cell activities at low ATP concentrations, a more sensitive microplate luminometer is required. Therefore, in this study, we developed an automated highly sensitive microplate luminometer that could perform reagent dispensing and bioluminescence measurement with a 96-well microplate within 10 min. ATP bioluminescence was detected by pressing a photomultiplier tube (PMT) against a microplate surface to seal the measured well with the light-receiving surface of the PMT. This enabled a high light collection efficiency and low luminescence crosstalk, defined as the intensity of stray light from an adjacent well. As a result, the ATP detection limit was 0.97 amol, and the luminescence crosstalk was 4.4 × 10-6 . Both values were one order of magnitude better than that of a typical microplate luminometer. In addition, the same gradient linearity of luminescence intensity against the ATP concentration was confirmed for both high and low ATP concentrations, and the dynamic range of our microplate luminometer was 106 . Overall, our findings demonstrated that our novel microplate luminometer may have wide application in biological sciences research.

A Rapid ATP Bioluminescence-based Test for Detecting Levofloxacin Resistance Starting from Positive Blood Culture Bottles.

Administering appropriate antimicrobial therapy as early as possible is important for rescuing bacteremic patients. Therefore, rapid antimicrobial susceptibility tests in positive blood culture specimens have been diligently sought. Adenosine triphosphate (ATP) bioluminescence-based methods have been used for rapid antimicrobial susceptibility tests. However, blood culture specimens have not been examined in many studies, possibly due to abundant intracellular ATP in blood corpuscles resulting in false-susceptible results. In this study, we developed a rapid ATP bioluminescence-based method for detecting antibiotic resistance starting from positive blood culture. To minimize background ATP originating from blood corpuscles, specimens were centrifuged and the supernatant diluted with broth, and an ATP-eliminating reagent was then added to the bacterial suspension at the beginning of incubation. This newly devised procedure reduced the background ATP by more than five orders of magnitude. In a pilot study using levofloxacin, no false-susceptible results were observed in 15 clinical specimens. Furthermore, the results indicated that the rapid method provided additional information about bacterial activities with high resolution, in contrast to the less-thorough findings with the conventional turbidity method. Therefore, our approach will contribute to the treatment of infectious diseases as a rapid antimicrobial susceptibility test.

A simple and highly sensitive spectroscopic fluorescence-detection system for multi-channel plastic-microchip electrophoresis based on side-entry laser-beam zigzag irradiation.

A five-color fluorescence-detection system for eight-channel plastic-microchip electrophoresis was developed. In the eight channels (with effective electrophoretic lengths of 10 cm), single-stranded DNA fragments were separated (with single-base resolution up to 300 bases within 10 min), and seventeen-loci STR genotyping for forensic human identification was successfully demonstrated. In the system, a side-entry laser beam is passed through the eight channels (eight A channels), with alternately arrayed seven sacrificial channels (seven B channels), by a technique called "side-entry laser-beam zigzag irradiation." Laser-induced fluorescence from the eight A channels and Raman-scattered light from the seven B channels are then simultaneously, uniformly, and spectroscopically detected, in the direction perpendicular to the channel array plane, through a transmission grating and a CCD camera. The system is therefore simple and highly sensitive. Because the microchip is fabricated by plastic-injection molding, it is inexpensive and disposable and thus suitable for actual use in various fields.

Far-ultraviolet absorbance detection of sugars and peptides by high-performance liquid chromatography.

A far-ultraviolet (FUV)-absorbance detector with a transmission flow cell was developed and applied to detect absorbance of sugars and peptides by HPLC. The main inherent limitation of FUV-absorbance detection is the strong absorptions of solvents and atmospheric oxygen in the optical system as well as dissolved oxygen in the solvent. High absorptivity of the solvent and oxygen decreases transmission-light intensity in the flow cell and hinders the absorbance measurement. To solve the above drawbacks, the transmission-light intensity in the flow cell was increased by introducing a new optical system and a nitrogen-purging unit to remove the atmospheric oxygen. The optical system has a photodiode for detecting the reference light at a position of the minus-first-order diffracted light. In addition, acetonitrile and water were selected as usable solvents because of their low absorptivity in the FUV region. As a result of these implementations, the detectable wavelength of the FUV-absorbance detector (with a flow cell having an effective optical path length of 0.5mm) can be extended down to 175nm. Three sugars (glucose, fructose, and sucrose) were successfully detected with the FUV-absorbance detector. These detection results reveal that the absorption peak of sugar in liquid phase lies at around 178nm. The detection limit (S/N=3) in absorbance with a 0.5-mm flow cell at 180nm was 21µAU, which corresponds to 33, 60 and 60µM (198, 360, and 360pmol) for fructose, glucose, and sucrose, respectively. Also, the peptide Met-enkephalin could be detected with a high sensitivity at 190nm. The estimated detection limit (S/N=3) for Met-enkephalin is 29nM (0.29pmol), which is eight times lower than that at 220nm.

Side-entry laser-beam zigzag irradiation of multiple channels in a microchip for simultaneous and highly sensitive detection of fluorescent analytes.

A simple and highly sensitive technique for laser-induced fluorescence detection on multiple channels in a plastic microchip was developed, and its effectiveness was demonstrated by laser-beam ray-trace simulations and experiments. In the microchip, with refractive index nC, A channels and B channels are arrayed alternately and respectively filled with materials with refractive indexes nA for electrophoresis analysis and nB for laser-beam control. It was shown that a laser beam entering from the side of the channel array traveled straight and irradiated all A channels simultaneously and effectively because the refractive actions by the A and B channels were counterbalanced according to the condition nA < nC < nB. This technique is thus called "side-entry laser-beam zigzag irradiation". As a demonstration of the technique, when nC = 1.53, nA = 1.41, nB = 1.66, and the cross sections of both eight A channels and seven B channels were the same isosceles trapezoids with 97° base angle, laser-beam irradiation efficiency on the eight A channels by the simulations was 89% on average and coefficient of variation was 4.4%. These results are far superior to those achieved by other conventional methods such as laser-beam expansion and scanning. Furthermore, fluorescence intensity on the eight A channels determined by the experiments agreed well with that determined by the simulations. Therefore, highly sensitive and uniform fluorescence detection on eight A channels was achieved. It is also possible to fabricate the microchips at low cost by plastic-injection molding and to make a simple and compact detection system, thereby promoting actual use of the proposed side-entry laser-beam zigzag irradiation in various fields.

Terahertz emission from surface-immobilized gold nanospheres.

Electromagnetic wave emission based on optical rectification at terahertz (THz) wavelengths was observed from surface-immobilized gold nanospheres (SIGNs) above a gold surface. Although the excitation wavelength is off-resonant with the localized surface plasmons, the THz emission field was observed to be approximately 4.8 times greater than that from a percolated gold thin film of 10 nm thickness. A theoretical calculation predicts that the light electric field is enhanced in the SIGN system, even at off-resonance wavelengths. The observed THz field amplitude was quadratic with the illumination light field, suggesting that the THz generation is due to a second-order nonlinear optical process.