Hiroshi Aoki (Research Scientist), the Measurement Technology Group (Leader: Hiroaki Tao), the Research Institute for Environmental Management Technology (Director: Koh Harada) of the National Institute of Advanced Industrial Science and Technology (AIST) (President: Hiroyuki Yoshikawa), developed an array spotter capable of dispensing an infinitesimal amount of liquid sample and of changing freely the intervals between the capillaries that serve as dispensing outlets.

In recent years, technologies for comprehensive analysis of a large number of biologically relevant materials, such as the mass spectrometry and DNA microarrays, have increasingly been used.  Further, technology has also been developed for pre-treatment of patients' samples for comprehensive analysis by using the above mentioned analytical equipment.  However, with regard to the present technology introducing a pre-treated liquid sample into the equipment, i.e. a "linkage technology", the rapidity is insufficient, handling of a small amount of liquid sample is difficult, and the dispensing precision is low.  Therefore, the overall performance of the comprehensive analysis has substantially been spoiled.

The researcher has developed an array spotter that can rapidly dispense nanoliter-level liquid samples, provided with a dispensing head bearing eight capillaries as dispensing outlets with adjustable capillary intervals. Thus, this is expected to help realize a more rapid and comprehensive analysis/diagnosis of a wider range of specimens based on DNA microarray or soft ionization mass spectrometry in fields of environment, biotechnology, and medical care.

A part of the results will be exhibited as "Highly Integrated Spotting Device for High-Throughput Analysis of an Infinitesimal Amount of Sample" at the AIST Open Lab held at the AIST Tsukuba on October 20 and 21, 2008.

The developed array spotter and its dispensing head bearing eight capillaries as dispensing outlets with adjustable capillary intervals.

The technology employed for a comprehensive analysis of biologically relevant materials has almost attained maturity.  Such technologies have increasingly been used not only in laboratory analyses of metabolites for drug discovery but also as a part of clinical assay including group medical examinations.  Technology for the pre-treatment of patients' specimens for the analysis has also advanced.  Laboratory automation technologies, such as automatic dispensing device and sample management system, are the key technologies employed for pre-treatment.  A desired amount of a desired liquid sample can efficiently and reliably be handled on a microplate.  However, the spotting technology employed to link a pre-treatment process and an analyzing process remains immature and lacks rapidity, and is not suitable for precise handling of small volume of liquid samples.  As a result, the performance of analytical equipment cannot fully be utilized, thereby substantially spoiling the efficiency of the whole comprehensive analysis.  Further improvement in comprehensive analytical technology is required for high-throughput screening of a number of samples collected for drug discovery or group heath examinations.

There exists a strong desire for the development of an evaluation/measurement method to implement the most appropriate risk management system for chemical compounds; therefore, the Research Institute for Environmental Management Technology has been developing a comprehensive analytical technology including environmental diagnosis.  Generally, such analytical technologies must be smoothly coordinated with sample pre-treatment devices.  In the conventional dispensing technology, when a liquid sample is spotted onto a substrate of analytical equipment from wells of microplate, a trade-off relationship exists wherein the rapidity counteracts the high precision/integration of the dispensing operation, and the overall performance of analytical method is impaired.  Therefore, considering that a new dispensing method is indispensable for the advancement of comprehensive analytical technology, we undertook the development of an array spotting device with high rapidity, precision, and integration.

We developed a mechanism in which intervals between multiple capillaries (used as dispensing outlets) can freely be changed.  This mechanism is compatible with all commercially available microplates, and the sample container and substrate that have different alignment specifications can be flexibly connected, thus enabling a rapid simultaneous dispensing of liquid samples.  The capillary intervals can be varied from 9 mm (maximum) to 0.9 mm (minimum) for an outer diameter of capillary of 360 µm.  A syringe pump is communicated to the capillary for suction/discharge of a liquid sample through the tip of the capillary in order to build a liquid-handling system.  By completely removing the air remaining in the liquid-handling system, the sample dispensing volume can freely be set so that it supports a wide range of small-scale to large-scale operations.  Moreover, nanoliter-level precise dispensing of an infinitesimal amount becomes possible.  We succeeded in developing a technology that can dispense an infinitesimal of liquid sample with high precision, integration, and rapidity.

In the conventional dispensing technology, a pipette tip, pin, needle, or inkjet head is used for the outlet port.  A pipette tip, pin, and needle are used mainly for dispensing or spotting between microplates.  If the specifications such as the size or spotting intervals of the sample substrate are different from those of the microplate, all the dispensing outlets cannot be used, thus spoiling the efficiency of the overall analysis operation.  Although the recently developed inkjet head-dispensing device can precisely dispense a small volume of a liquid sample, suction is technically impossible.  Therefore, the liquid that is to be dispensed must be filled into the dispensing head in advance.  Accordingly, the inkjet head is not suitable for treating a number of liquid samples on a microplate.  Using our technology, several liquid samples can simultaneously be suctioned from the microplate and then directly dispensed onto the substrate at desired intervals by changing the capillary intervals.

The newly developed technology can deal with a smaller quantity of liquid samples as compared to that supported by the conventional technology, and the dispensing precision is improved.  In the conventional variable-capacity pipette tip system, 500 nL is the minimum dispensing amount, and the relative error in the dispensing amount is 5-8% (25-40 nL) for 500 nL.  In the pin system, which employs a conventional fixed-capacity spotting technology, the minimum dispensing amount is of the order of a few nanoliters, although the relative error is considerably large, e.g. several tens percents.  The needle system is intermediate in terms of functional properties.  In contrast, the developed array spotter has a minimum dispensing amount of 10 nL and a relative error of 2.7% (0.54 nL) for a dispensing amount of 20 nL (Table 1).  Using this technology, the quantity of a sample necessary for each analysis is reduced; therefore, more various tests can be performed repeatedly.  Thus, a more highly precise analysis/diagnosis can be achieved.

Table 1. Evaluation of dispensing precision

a) The values of volume error were derived from those of dispensing volume and relative error.

We attempted to apply the array spotter to soft ionization mass spectrometry, which is currently attracting attention as a high-throughput analytical method.  The Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOFMS) method uses approximately 500 nL of liquid sample per spot, based on the current dispensing technology.  The spot diameter is estimated to be approximately 2 mm, and the spot interval is approximately 4–5 mm, considering the spreading of sample on the substrate.  These values are apparently too large in comparison with the 100–300µm laser irradiation diameter of the usual MALDI-TOFMS, thereby suggesting that there exists a wasteful space on the substrate that is not used for the measurement.  We confirmed that the substrate and analytical equipment can be downsized, because the sample dispensation is performed at narrower intervals in our device as compared to that of conventional spotters (Fig. 1).

Figure 1. A photograph of an 8 x 8 spot array on a sample substrate (dispensed volume: 10 nL, spot interval: 1 mm) supported by a commercially-available sample substrate (spot interval: 5 mm).

The internal standard mass calibration method is generally used in MALDI-TOFMS wherein a reference compound of a known mass number is mixed with the liquid sample.  However, when a sample of unknown mass number/concentration is examined in a proteome or metabolome analysis, optimization of the reference compound is difficult and it becomes a hindrance to high-throughput analysis.  In order to solve this problem, the external standard mass calibration method is devised, wherein a reference compound and liquid sample are separately applied onto the sample substrate, although the mass precision is significantly lowered upon increase in the distance between the two spots.  Using the newly developed spotter, high-precision external standard mass calibration becomes possible by dispensing the reference compound onto the neighborhood of the sample spot within an extremely short distance.  Therefore, the spotter is found to be effective in improving the rapidity and analytical performance of MALDI-TOFMS.  Because the existing mass analytical equipment is a product of compromise designed to fit the current dispensing technology specifications, the inherent performance cannot fully be utilized.  When the developed array spotter is used, the present sample pre-treatment specifications are satisfied, and a compact and efficient substrate and instrumentation is realized at the same time.

Drawing on the distinctive characteristics of our device, namely, high-precision, high-integration, and rapid dispensation of an infinitesimal amount of a liquid sample, the array spotter is expected to significantly contribute toward the development of tailor-made medicines based on comprehensive genetic diagnosis technology, for example, by using DNA microarrays.  Further, because a sample substrate can be promptly and easily prepared for use in mass spectrometry, it will become possible to rapidly dispense a small amount of a liquid sample, including biologically-relevant materials having unstable natures.

We will make further efforts in broadening the applications of this technology and minimizing the dispensing amount and capillary intervals.  Simultaneously, we will aim for the commercialization of the technology in collaboration with the companies that will be interested in the array spotter.