Automated Analysis System of Biomolecular Interaction byIndex-Matching Fluid-Mediated Fluorometry
By Satoshi Sugiyama *, Jun Nishina *2, Ken Tsukii *, Shinichi Taguchi*,
Kenichi Kimura*, Toru Takahashi * and Jie Xu *In this post-genome era, analysis of the functions of proteins and other biomolecules is becoming an important area of study, creating a need for highly versatilesystems capable of the high-sensitivity measurement and analysis of biomolecular interactions. In the research reported here, we have developed a system for the automatedanalysis of biomolecules by “fluid-mediated fluorometry”, which is a new technique of measurement. Due to the approximate doubling of measurement sensitivity and the anti-drying effectoffered by this measurement technique, we have achieved real-time measurement of minutesamples. Using this system we have evaluated interactions between synthetic peptides and proteins and agonist measurement of yeast as a biomolecular tool, confirming its efficacy as a highversatility, high-throughput technique for biomolecular interaction analysis.

ABSTRACT1.

INTRODUCTION

Proteins which constitute genetic products are directly
involved in the life and living of the human body and aretargets for the majority of drug actions. Today, withgenome analysis completed for many species, aminoacid sequences of proteins have been predicted from thecoding genes, however it is impossible to identify theirfunctions by genetic information alone since proteins arealways modified after translation by many biochemical
processes such as phosphorylation, glycosidation, acetylation and methylation. Thus the detection and functional
analysis of proteins have become an important area of
research that aims to tie these masses of genomic information to the design of drugs and tailor-made therapies.
To this end, peptides that are low in molecular weightand easy to synthesize are extensively used to construct
the libraries of peptides for approaches to identification
and analysis of casual proteins of diseases and improvement of the screening efficiency for lead compounds that
constitute bottlenecks in terms of the time and money
required to develop drugs.
Accordingly there is an urgent need for a highly versatile system capable of high-throughput, high-sensitivity
measurement and analysis of biomolecular interactions.
In the present study, we have developed a system for the
automated analysis of biomolecules--from sample acquisition to reaction and measurement--by “fluid-mediatedfluorometry”, a novel technique of measurement (see* Bio Device Group, Production Engineering Center, Production
Technology and Engineering Division.
*2 Yokohama Lab., R&D Div.

Figure 1

Automated analysis system.

Figure 1).

2.

EVALUATION OF FLUID-MEDIATEDFLUOROMETRY

2.1 Principle of Fluid-Mediated Fluorometry
In fluorometry using an optical detection head, clearance
must be provided between the detection head and theobject of measurement in order to prevent contamination,and the Fresnel reflection loss, etc. of the excitation lightand fluorescent light at the refraction interfaces between
the tip of the optical detection head and the air, andbetween the object of measurement and the air constitutefactors in the decrease of the fluorescent acquisition efficiency.

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Automated Analysis System of Biomolecular Interaction by Index-Matching Fluid-Mediated Fluorometry

Further, most of the organic samples usually exert their
physiological functions in a liquid endogenous environment. Loss of function due to drying during the detection
becomes a problem.
Optical ﬁber

Index matching ﬂuid
Sample to be measured

Micro plate

Figure 2

Fluid-mediated fluorometry.

Our fluid-mediated fluorometry technique (see Figure2), is characterized by the space between the opticaldetection head and the object of measurement being
filled with droplets (hereinafter referred to as fluid) thereby
eliminating the layer of air that contributed to a decrease
in sensitivity as well as to drying. By selecting as the fluida material that has an index of refraction close to that of
the optical detection head or the object to be measured it
becomes possible to achieve high-sensitivity measurement. And if a fluid having low affinity with the measurement samples is used, contamination does not occur
even if the detection head comes into contact with thefluid. If, in addition, the fluid is non-volatile, an anti-evaporation effect on the biological samples can be expected.
2.2

Numerical Study of the Role of Fluid Mediation in
Improving Sensitivity
We carried out a simple numerical study of two factors in
the improvement in sensitivity in fluid-mediated fluorometry, namely (1) reduction in the Fresnel reflection loss, and(2) enlargement of the numerical aperture (NA).
2.2.1 Reduction in Fresnel Reflection LossIf light impinges on an interface at which the index ofrefraction changes discontinuously, part of the advancing
power is reflected. The reflectance is dependent on theindex of refraction, an incidence angle, and polarization ofincident light. However, as long as an incidence light islimited to perpendicular incidence, it will not depend onpolarization. In this case, the Fresnel coefficient of perpendicular incidence (power reflection coefficient) may beshown as� �� �� � ＝ � �� � � � �
� �
��

(1)

Here, n1 and n2 represent the indexes of refraction of
medium 1 and 2, respectively; r indicates the strength
ratio of catoptric light to incident light, and r2 expresses

the ratio of power. Thus, for example, when n1 is 1.515
and n2 is 1.000, r2 will be 0.0419, and when laser light iscoupled to an optical fiber with no coating, a reflection
loss of approximately 4 % is unavoidable.
Even in fluorometry using microplates, Fresnel reflection constitutes a major obstacle. If we consider a case inwhich excitation and received power are both carried out
by an optical glass fiber probe, the excitation light will
undergo a loss of approximately 6 %--about 4 % at thefiber-air interface and about 2 % at the air-sample interface. This will be analogous for the fluorescent light that
under the irradiation of excitation light is emitted to
impinge on the fiber, so that added together more than
10% of the overall sensitivity will be lost due to interface
reflection. When components other than perpendicularincidence are also included, the loss becomes evengreater. In fluid-mediated fluorometry, the intervening
gaps are filled with oil having a high index of refraction sothat, in effect, the hetero interfaces are only from opticalfiber to sample and back and total reflection loss can beheld to less that 1 %. This is advantageous not only interms of sensitivity, but also in eliminating stray light in themeasurement of faint fluorescent light.
2.2.2 Enlargement of Numerical Aperture (NA)
Effect of the fluid on enlargement of the numerical aperture (NA) was then investigated. Just as in the oilimmersed objective lens of a microscope, where the gap
between the sample and the objective lens is filled withoil, fluid-mediated fluorometry uses an oil having an index
of refraction much higher than that of water or glycerin,
making it possible to obtain a larger numerical aperture.
D coreMedium-3: ﬁber
�3＝1.515

R2
R1

θ3

Medium-2: air or oil
�2＝1.000
or 1.515

L2
θ2

L1

θ1
Medium-1: water
�1＝1.333

Figure 3

Effect of enlargement of numerical aperture by fluidmediation.

Figure 3 shows a cross-section of the apparatus for
fluid-mediated fluorometry using microplates. The fluorescent light emitted from the sample under excitation isrefracted at the interface and detected by the optical fiber.
The fluorescent light is emitted isotropically in all directions, and polarization may be taken as negligible. For
simplicity it is assumed that the fluorescent samples arepoint light sources which have no size and are positioned
vertically under the optical fiber. Medium 1 is a fluid that
contains the sample, with an index of refraction of 1.333,
equal to that of water. Medium 2 is the intervening layer
which, in the fluid-mediated method is assumed to be sili-

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Automated Analysis System of Biomolecular Interaction by Index-Matching Fluid-Mediated Fluorometry

cone oil having an index of refraction of 1.515, but in theconventional measurement method is taken to be an air
gap with an index of refraction of 1. Medium 3 is the index
of refraction of the optical fiber core, or 1.515 in this case.
The angles of refraction at the interface (θ1 - θ3) arelinked by Snell’s Law so that θ3 and R2 may be represented using θ1 as
�

� ��
�
����� ����������������������� � �������� ���

(2)

�

�
�
� ��
��
� ��������������������������������
����
���
��
�� ������ � ������������
�
�
� �
��
�

(3)

wider L2 is, the narrower will be the solid angle at whichlight can be detected, the graph as a whole shows a tendency to descend to the right. It can be seen that in acase in which a specialty fiber of high NA is used for theprobe, the angle at which light can be detected will beapproximately an order of magnitude greater that with theconventional multimode fiber.
To estimate the ratio of enlargement of photodetection
sensitivity due to the fluid, we find I1max with respect to thecases in which medium 2 is oil and air respectively, anddefine the value of the ratio as the figure of merit (FOM):

Here, n1 ~ n3 represent the index of refraction of eachmedium, respectively; θ1 stands for the incident angle oflight from medium 1 to medium 2, θ2 and θ3 for the angles
of incidence and refraction from medium 2 to 3, respectively. R1 and R2 indicate the distance between the lightbeam and the central axis of the optical fiber core when it
passes through the medium interface. L1 expresses thedepth of fluorescent light source within medium 1, and L2
shows the gap between the interface of medium 1, 2 andthe optical fiber (medium 3). The conditions under which
the light beam is coupled to the optical fiber are such thatthe point of intersection of the light beam and the fiber
end face is within the core, and the angle of incidence isless than the NA of the fiber, that is, both�� ����� ���≦����������

□ Ｓpecialty ﬁber

Sensitivity（rel.）○ Ｃonventional ﬁber

0.15
0.10
0.05

0

0.2

0.4

0.6

L 2 （mm）

Figure 4

FOM

1.0

0.5

0.0

0

0.2

0.4

0.6

L 2（mm）

Sensitivity figures of merit (using specialty fiber).

(5)

0.25

0.00

1.5

Figure 5

are satisfied. Note also that the well aperture of the microplate shall be sufficiently large, and that there shall be noloss due to blocking of light by the brim of the well.
Fluorescence sensitivity shall be proportional to I 1max
which is obtained by integrating, after solid angle conversion, the maximum value of �θ1 that satisfies the conditions of Equations (4) and (5) simultaneously, with respect
to fluorescent light source depth L1.
Figure 4 shows I 1max when gap L 2 at the interfaces
between the fiber and media 1~2 is changed. Since the

0.20

(6)

2.0

(4)

��
� �����
������
��� ���≦
��������������
���������

������

Dependence of measurement sensitivity on gap
thickness.

Figure 5 is a graph plotting FOM versus gap L2 whenusing a special optical fiber. It was found that a sensitivity
merit of approximately 1.5 folds could be obtained, largely
independent of changes in L2.
Based on the discussion above, it can be seen thatthrough the reduction in Fresnel reflection loss and NA
enlargement, an improvement in sensitivity of up to double can be anticipated.
2.3

Implementation of Fluid-Mediated Fluorometry

2.3.1 Samples and Fluid Conditions
As shown in Figure 6, we brought the optical fiber headthat emits the excitation light down from above into proximity with a well that has been filled with a fluorescentreagent and the fluid, and obtained the detection intensityof the resultant fluorescence and reflected light. The fluorescent reagents were diluted with N, N, N’, N’-carboxytetramethylrhodamine (TAMRA) and used. The well used
was of substantially cylindrical configuration with a diameter of 1.4 mm, having 8 µL of fluorescent reagent on
which index-matching silicone oil was dripped. The measured value was taken as the fluorescent intensity at the
point at which the optical fiber head approached to a
position of 0.3 mm with respect to the fluorescent pigment
surface.

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Automated Analysis System of Biomolecular Interaction by Index-Matching Fluid-Mediated Fluorometry

12000

Intensity FL（digit）

Multimode optical ﬁber

40000

11000

Backscattered light

10000
9000

35000

Head aproachingdirection

8000
7000

30000

Fluorescence

6000

25000

Contact position tointervening liquid

5000
4000
-1

-0.7

-0.4

-0.1

0.2

0.5

Intensity BS（digit）

Index matching ﬂuid（2 µl）

20000

Head position（mm）

Figure 8
Fluorescent solution（8 µl）

Figure 6

Schematic of conditions for evaluation of fluid-mediated fluorometry.

2.3.2 Fluorescence Optical System
Figure 7 depicts the fluorescence optical system used in
fluid-mediated fluorometry.
In order to support high-sensitivity fluorometry, a monochrome laser source was used for excitation light, with aspectroscope using dichroic mirrors, bandpass filters and
PMTs (photomultiplier tube) for detection. The sensing
optical fiber, for purposes of high-sensitivity fluorometry,
had a large NA and was designed based on MFD (modefield diameter) suited to the requisite level of measurement gap resolution, and the lens system was optimized
to match this.
In addition to being designed with the capability of polychromic spectrometry, measurement of fluorescence pigments for a variety of applications can be supported bychanging the excitation light source and optical elements.
Measurement of backscattered light is also supported toallow data compensation through status recognition anddetection of status error. In this way it is also possible forthe optical fiber head to approach or come into contact
with the fluid plane, as recognition of the measurement
status.

Fluid-mediated fluorometry monitored 2-channel signals.

one well. The main factors affecting reflected light arecaused by: (1) the end face of the optical fiber, (2) the
fluid plane of the sample measured; and (3) the reflectionof excitation light at the plate surface. There is a gradualincrease in the reflection light signal as the tip of the optical fiber approaches the well, but since, if it comes into
contact with the fluid, the reflection due to factors (1) and(2) above decreases significantly, detection sensitivity will
decline.
The fluorescence signal also increased as the opticalfiber approaches, but unlike the reflected light, when it
comes into contact with the fluid, the intervening air gap
disappears, so that it rises significantly.
2.4

Effect of Improvement in Sensitivity by FluidMediated Fluorometry

2.4.1 Improvement in Measurement Sensitivity
In order to obtain direct confirmation of the effect of sensitivity improvement achieved by fluid-mediated fluorometry, we first measured without using fluid, and carried out
comparative measurements in that condition with fluid
supplied. As a result measurement sensitivity was

2.3.3

Results of Fluorometry Monitored by
Fluorescence and Reflection Channels
Figure 8 shows an example of results of measurement of
PMT for
FL2Band pass ﬁlter
PMT for
FL1

Laser source
Dichroic mirror
Lens

Excitation
light

Detected light

Optical ﬁber probe

Figure 7

With silicone oil

Without silicone oil
8000
Intensity（digit）

PMT for
BS1

PMT for
FL3

7000
6000

With silicone oil

5000
4000
3000
2000

Without silicone oil

1000※ Laser source and all opticalelements are replaceable forparticular ﬂuorescence bands.
※ Backscattered light can also be
detected for status monitoring.

Optical system used in fluid-mediated fluorometry.

0

0

0.1

0.2

0.3

0.4

0.5

0.6

TAMRA concentration（μM）

Figure 9

Effect of improvement in measurement sensitivity by
fluid-mediated fluorometry.

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Automated Analysis System of Biomolecular Interaction by Index-Matching Fluid-Mediated Fluorometry

improved by two folds (see Figure 9), agreeing with theresults of the numerical analysis presented above. This isthought to be due to the reduction of Fresnel reflection
loss in the refractive index interface and by the increase in
the photodetection NA due to fluid mediation.
120
Index matching ﬂuid- mediated measurement

80

30000

60

20000
Conventional plate reader

10000-10000

40
20

0
0

0.1

3.

100
Relative intensity
by plate reader

Intensity FL (digit)

40000

0
0.3
-20

0.2

evaluation difficult. In contrast, it was found that when anon-volatile fluid was used, drying was prevented, stablemeasurements could be taken even for minute samples,and changes in the properties of the object of measurement could be determined over long periods in real time.

TAMRA concentration（ｎM）

Figure 10 Detection of fluorescent signal using low-concentration samples.

Figure 10 shows the result of measurements carried out
at low levels of concentration. It was possible to obtainadequate measurement provided the concentration wasin the order of 0.1 nM. To detect feeble signals from particularly low-contration samples requires not only improvements in the efficiency of condensation of the fluorescentlight but also reduction in background noise. Since interface reflection of excitation light is a major factor in background noise, it is a major advantage in terms of detecting feeble fluorescence that fluid-mediated fluorometry
enables the reflected light together with its deviation to be
reduced for each detection event.
Anti-drying Effect of Samples by Fluid-mediatedFluorometry
We also conducted a measurement evaluation withrespect to the anti-drying effect, which is a further advantage of fluid-mediated fluorometry (see Figure 11). The
well had a deep-hole configuration measuring 1.4 mm indiameter and 4 mm in depth, but even so, when no fluid
was used there was a marked drop in detected fluorescent intensity due to drying. This makes it impossible todisregard the passage of time after sorting or the effect ofthe measurement procedure, and makes quantitative

DEVELOPMENT OF AUTOMATEDANALYSIS SYSTEM AND ITSAPPLICATION TO BIOMOLECULAR
MEASUREMENT

3.1 Automated Analysis System
In accordance with the concept of an automated analysis
system, we have developed a system (see Figure 12)
capable of performing the series of operations--fromacquisition of multiple samples through sorting, mixing,
and real-time reaction measurement, to the following processes for the purpose of measurement (cleaning anddrying of the optical fiber tip, replacing of the pipette tip,etc.)--in a fully automated manner.
Rotary tool selector
PipetteMeasuring headIndex matching ﬂuid dispenser
Tip replacing unit
Tip rack
Sample plate

Micro plate
Thermal and vibratory
controller

2.4.2

1.2
With silicone oil

Intensity（digit）

1
0.8
0.6
0.4

Without silicone oil

0.2
0

0

5

10

15

Time after pipetting（h）

Figure 11 Anti-drying effect by fluid-mediated fluorometry.

Cleaner and drier

Figure 12 Core units of the automated analysis system.

3.2

Evaluation of Peptide-Protein Interactions

3.2.1 BackgroundA system that could provide simple and efficient detectionof proteins, and was also capable of analyzing their physical and chemical properties and their functional nature
would be enormously useful. Dr. Mihara’s Group at theTokyo Institute of Technology has focused on the configuration recognition properties of proteins, designed andsynthesized peptides with a variety of 2- and 3-dimensional structures, and is engaged in developing a novel
assay method for detection of protein phosphorylation as
well as constructing protein chip for detecting and analyzing proteins 1)〜8）.
Peptides are of lower molecular weight than proteins,and it is possible to design and synthesize many different
2- and 3-dimensional molecular configurations, including
α-helix, β-sheet, loop structure and so on. By means ofprotein fingerprinting using fluorescent-modified peptide
arrays, it is possible to characterize large numbers of pro-

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Automated Analysis System of Biomolecular Interaction by Index-Matching Fluid-Mediated Fluorometry

surements were made for each well individually the rate ofmeasurement was 1 well per second, but scanning of the
optical fiber head enables measurement of 50 wells per
second.
4
3.5
3

L8K6

2.5
⊿I/I0

teins. In addition to the detection of specific proteins, thismethod also holds promise of functional analysis of proteins in the form of analysis of the bonding characteristics
of proteins, and the investigation of peptide ligands thatcan control the function of proteins. In fact Dr. Mihara’s
Group, taking as a model the calmodulin-calcineurin(CaM-Cn) system shown in Figure 13, is configuring systems such as one for investigating a peptide ligand thatcan control the dephosphorylation activity of Cn by bonding with CaM 9).
In the following section we shall discuss an evaluation,
carried out jointly with Dr. Mihara’s Group, that utilized theadvantages of fluid-mediated fluorometry is the stable,
high-sensitivity measurement of the status of solutions in
its applicability to the functional analysis of proteins.

2
1.5
1
0.5

L8K4E20-0.5 0

0.2

0.1

-1

0.3

0.4

0.5

CaM concentration（mg/ml)

Figure 15 Evaluation of magnitude of peptide-protein binding.

Cell
Cell
cytoplasm
Dephosphorylation

NUC

Phosphorylation

Ｔ-Cellactivation

Production

Figure 13 Immune signaling system using calmodulin and calcineurin.

3.2.2 Evaluation of Peptide-protein Interactions(1) Measurement of CaM- L8K6 combination
First of all, to evaluate the suitability of fluid-mediated fluorometry for measuring combination of proteins and peptides, we conducted evaluations of the binding between
CaM and L8K6 peptide, which is already known, and of
the binding to L8K2E4 peptide, which has low combination ability.
4 µL each of 2 µM peptide and a CaM solution with different concentration levels were mixed and sorted, andfluorescent intensity was measured by fluid-mediated fluorometry.
As shown in Figure 14, the well has an aperture for the
fluid above the portion for holding the sample. Since mea-

Figure 15 shows the results of the measurements. For
L8K2E4 peptide, which has low combination ability with
CaM, virtually no increase was observed in fluorescentintensity, but for L8K6 peptide, an increase in fluorescentintensity was observed as CaM concentration increased.
Under conditions of high CaM concentration measurement discrepancies occurred attributable to differences in
the amount of sorting due to adhesion to the pipette tip,
but the curve to the extent of saturation could be fitted tothe binding constant Ka of 6.8 x 106 M-1 between CaM andL8K6 peptide, confirming that it is possible to evaluate
binding between protein and peptide.
(2) Matrix evaluations of peptide-protein interactions
Next, in order to evaluate its applicability to protein analysis using the peptide library, a matrix evaluation was carried out using several peptides and proteins. As shown in
Table 1, we used a total of five standards--four proteins
and a buffer solution--for evaluations of four types of synthetic peptides, and made a relative evaluation of theincrease in fluorescent intensity due to binding.
Concentrations were 3 µM for the proteins and 1 µM forthe peptides; the conditions of well and capacity weresimilar to those of the previous section.
Table 1

Measurement matrix elements for evaluation of
peptide-protein interactions.
Synthetic peptide（1 µM）
TAMRA- TAMRA- TAMRATAMRAL8K6
L8K2E4 L6A2K4E2 A8K2E2

φ3 mm
φ3mm
Calmodulin
（CaM）

4.5
mm
4.5mm

5
mm
5mm

φ1.5 mmFigure 14 Schematic structure of the well for peptide-protein
measurement.

Alpha amylase（α-ami）
ProteinProtein kinaseA（3 mol/l）（PKA）
Insulin（Ins）
Buffer

○

○

○

○

○

○

○

○

○

○

○

○

○

○

○

○

○

○

○

○

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Automated Analysis System of Biomolecular Interaction by Index-Matching Fluid-Mediated Fluorometry

3.5
3

L8K6

2.5

L8K2E4L6A2K4E2⊿ I/I02

A8K2E2

1.5
1
0.5
0
-0.5

none

CaM

�-amy

PKA

Ins

Protein（3 µM）

Figure 16 Results of matrix measurement of peptide-protein
interactions.

Figure 16 shows the results. With respect to L8K6 peptide, binding with CaM was the strongest, and binding
also occurred with α-amylase and protein kinase A but
not with insulin. These results suggest that the magnitudeof binding can be evaluated by fluorescent intensity. With
respect to L8K2E4 peptide, limited to the protein standards used here, we may say that binding occurred onlywith α-amylase, suggesting that specific binding can bedetected. These results indicate that it is possible, byusing multiple species of synthetic peptides, to screen
ligands acting with specific proteins, and by means ofprotein chips using synthetic peptides, to detect proteins.

human receptors and its application to high-throughput
drug design screening.
In the following section we report on measurement ofyeasts for agonist detection by fluid-mediated fluorometry
carried out in conjunction with Kobe University.

3.3

3.3.2

Development and Evaluation of Ligand Screening
System using Yeast

3.3.1 BackgroundOn the cell membrane of the a-type cells of the yeast,
there are G protein conjugate receptors (GPCRs) known
as Ste2p, to which binds α-factor, a binding factor secreted by α-type cells. Dr. Kondo’s Group at Kobe University,
one of the participants in the present development work,
took GPCRs as the molecular target for drug design andare developing a ligand screening system utilizing signaltransmission of the a-type cells of the yeast.
By means of genetic methods, the target receptors
were expressed on the yeast cell surface and peptide
ligands were presented on the yeast surface. By transfection of library genes, different types of peptide ligands
could be presented on the various yeasts. Whenever in acell presented peptide ligand bound to the expressed
receptors as an agonist, signaling would be induced within the cell. At the final stage of the signaling pathway,
under the control of a FUS1 promoter whose transcription
activity was induced, reporters genes such as fluorescentprotein were expressed which in turn make more change
in the profiles of yeast genes so that positive agonists
could be screened (see Figure 17). That is, the yeasts
expressing positive ligands give off fluorescent light andare easily detected or separated, giving promise of an
extremely effective method of large-scale screening.
Dr. Kondo’s Group is developing a cell system that is
optimally suited to this screening. Especially, they are
working towards construction of such a system using

Figure 17 Screening system using EGFP.

Evaluation by Fluorescent Detection using
Peptide-presented Cells
The yeast cells were present dispersed within a medium,and since they settled to the bottom, a measurement
method was adopted in which 0.5 µL of a yeast cell suspension was sorted in a conical well as shown in Figure
18, approximately 10 µL of the fluid (index-matching silicone oil) was dripped from above, and the optical fiberhead was scanned 2-dimensionally. Analysis of the 2dimensional data thus obtained yielded readings of fluo-

0.5 mm

φ2 mm

Figure 18 Measurement of unevenly localized objects using
planar scanning head.

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Automated Analysis System of Biomolecular Interaction by Index-Matching Fluid-Mediated Fluorometry

rescence values for the cell portion.
We used a mutant of Saccharomyces cerevisiae
BY4741 [MATa his3∆1 leu2∆0 met15∆0 ura3∆0 bar1∆::LEU2
far1∆::kanMX4 sst2∆::AUR1-C Pfus1-FUS1-EGFP-HIS3］as theyeast host (hereinafter referred to as IMGH-8) in our system. Screening sensitivity was improved since this strain
is deficient in the following inhibitory protein of targeted
signaling as well as the proteins involved in other signaling pathways.
1. The Bar1p protease gene for degradation of α-factor
has been destroyed.
2. The Sst2p gene whose protein product impedes thesignaling through inhibiting separation reaction ofheterotrimer G-protein which works with the receptors binding to α-factor, has been destroyed.
3. The Far1p gene whose protein product will be activated by phosphorylation with the expression of
Fus1p at the final step of signaling and inhibit cell
cycle progress, has been destroyed.
As a high-expression reporter plasmid, a multi-copy
type pMHRS-pFUS1 which expresses Fus1-EGFP under
FUS1 promoter was used. pUESC α -FLO42 which
encodes and presents α-factor to the cell surface under
GAL1 promoter was used for presentation of α-factor, andan empty vector pESC-URA was used as a negative control. Through transfection of these plasmids into the hostcells, IMGH-8/pESC-URA and IMGH-8/pUESCα-FL042
cell clones were obtained. These clones were then cultured in an SD-Ura medium. Fifteen hours in an SG-Ura
medium, cells were started to amplify. To obtain a cellsuspension with an intensity of OD600 = 10, cells werediluted and measured at various phases.

Figure 19 shows the results for measurement of IMGH8/pESC-URA and IMGH-8/pUESCα-FL042. At cell counts
of approximately 103 or more a clear difference could beobserved. These results indicate that presentation on thecell surface is extremely useful in the study of ligand binding, and that resultant fluorescence of EGFP can bedetected by our system.

4.

CONCLUSION

We have developed a novel method called fluid-mediatedfluorometry to measure minute biomolecular interactions
using proteins and peptides as the main targets. This has
made it possible, by means of a fundamental analysis
using fluorescent pigment, to achieve approximately double the sensitivity together with an anti-drying effect, andto obtain real-time measurements of minute fluorescentsamples while in solution. We have also developed ahighly versatile automated analysis system to carry out
this measurement, and have confirmed its efficacy by
evaluating biological contents.

5.

ACKNOWLEDGMENTS

This research was supported in part by Grant-in-aid fromthe Kanto Economics and Business Bureau for 2004-05.
We thank Dr. Hisakazu Mihara of the Dept. of Bioscienceand Biotechnology, Tokyo Institute of Technology Schoolof Graduate and Dr. Akihiko Kondo of the Dept. ofChemical Science and Engineering, Faculty ofEngineering, Kobe University for their copious guidanceand cooperation.

30000

Intensity（digit）

25000
20000
15000

REFERENCES

IMGH-8/pUESC�-FLO42（◆）
IMGH-8/pESC-URA（◇）

10000
5000
0
-5000
1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

Number of cells

Figure 19 Evaluation of IMGH-8 with peptides presented on thesurface by detection of fluorescence.

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