Fluorescence detection in (sub-)nanoliter microarrays

Fluorescence detection in (sub-)nanoliter microarrays L.R. van den Doel, M.J. Vellekoop , P.M. Sarro , S. Picioreanu , R. Moerman , J. Frank , G. van Dedem , K.T. Hjelt , L.J. van Vliet, and I.T. Young DIOC-IMDS,  Pattern Recognition Group, Faculty of Applied Sciences, Lorentzweg 1, Delft University of Technology, NL-2628 CJ, Delft, The Netherlands, phone: +31-15-278-1416, fax: +31-15-278-6740, Electronic Instrumentation Laboratory, DIMES,

Electronic Components, Technology, and Materials, DIMES,  Bioprocess Technology Group email: fL.R.vandenDoel,I.T.Young,[email protected]

ABSTRACT

The goal of our TU Delft interfaculty research program is to develop intelligent molecular diagnostic systems (IMDS) that can analyze liquid samples that contain a variety of biochemical compounds such as those associated with fermentation processes. One speci c project within the IMDS program focuses on photon sensors. In order to analyze the liquid samples we use dedicated microarrays. At this stage, these are basically miniaturized micro titre plates. Typical dimensions of a vial are 200  200  20 m3 . These dimensions may be varied and the shape of the vials can be modi ed with a result that the volume of the vials varies from 0:5 to 1:6 nl. For all experiments, we have used vials with the shape of a truncated pyramid. These vials are fabricated in silicon by a wet etching process. For testing purposes the vials are lled with rhodamine solutions of various concentrations. To avoid evaporation glycerol-water (1 : 1; v=v) with a viscosity of 8:3 times the viscosity of water is used as solvent. We aim at wide eld-of-view imaging at the expense of absolute sensitivity: the eld-of-view increases quadratically with decreasing magni cation. Small magni cation, however, implies low Numerical Aperture (NA). The ability of a microscope objective to collect photons is proportional to the square of the NA. To image the entire microarray we have used an epi-illumination uorescence microscope equipped with a low magni cation (2:5  = 0:075) objective and a scienti c CCD camera to integrate the photons emitted from the uorescing particles in the solutions in the vials. From these experiments we found that for this setup the detection limit is on the order of micromolar concentrations of

uorescing particles. This translates to 108 molecules per vial. Keywords: microarrays, uorescence microscopy, wide eld imaging

1. INTRODUCTION

One of the most widely exploited elds of research during the last decade is that of combinatorial chemistry. The stated goal of this approach has been to drastically reduce the time and cost that is required for obtaining new lead compounds. Currently, many samples are tested against a large number of targets in microarrays for high-throughput screening. In the future, this technology will lead to a miniaturized lab-on-a-chip. At this moment, however, there are still many challenges to be solved:

 New types of chemistry need to be developed, because the possible targets need to be very sensitive and selective for high-throughput screening.  Liquid handling needs to be modi ed to the picoliter range. Ultra small volumes must be fast and automatically dispensed in (sub)-nanoliter vials in an accurate, precise, and reproducible manner.

M. Ferrari (eds.), Micro- and Nanofabricated Structures and Devices for Biomedical Environmental Applications II, Proc. SPIE, Progress in Biomedical Optics, vol. 3606, 1999, 28{39.

28

 A lab-on-a-chip is more than just a miniaturized micro titre plate: microarrays will contain vials with built-in

intelligence, i.e. integrated actuators and sensors  Many ( uorescent) signals can be simultaneously generated. Therefore, state of the art detectors and detection methods are necessary in order to perform quantitative analysis.  High-throuphput screening generates enormous amounts of data. In order to extract useful information, it is essential to develop special data analysis systems. Furthermore, data interpretation systems must be developed to derive new insight into the underlying biochemical principles.

The IMDS program aims to nd solutions for the challenges listed above. The goal of this research program is to develop systems that can analyze liquid samples that contain a variety of biochemical compounds such as those associated with fermentation processes. In contrast to most of the research done in this eld, our program does not focus on applications related to DNA. One of the main products obtained from human donor blood is Human Serum Albumin (HSA). Due to the risk of transmission of viral diseases, such as Hepatitis and AIDS, there is a strong need for an alternative source of human albumin. Unfortunately, human blood plasma is a very rich source of HSA ( 65 g=l) and the current price of HSA is low ($ US 2 ; 3 =g). This will make it very hard to replace it through any recombinant organism. Pichia pastoris, which is a yeast strain, produces HSA under a methanol-oxidase promotor. The expression of HSA contains contaminants, which must be removed during puri cation. The objective of this project is to nd correlations between quantities measured in the fermentation tank and the quantity and the quality of the HSA produced. The primary goal is to increase the productivity of HSA under the following constraints. First, the obtained quantities may not be puri ed at extremely high cost, and second, the product must be very pure, because it is normally administered in relatively high doses. In order to optimize the production under the given constraints, high-throughput screening is necessary: a sample that is taken from the fermentation tank will be tested against several targets. In order to analyze such a liquid sample, dedicated microarrays will be developed. In this stage of the project the microarrays are just miniaturized micro titre plates with a volume on the order of one nanoliter. The fabrication and design parameters of the microarrays will be described in section 2.2. In the future, the microarrays will be equipped with sensors and actuators. In each vial a sensor that measures the injected volume in the vial can be integrated. Other examples of sensors are temperature or pH sensors. Furthermore, optical waveguides might be integrated to direct the excitation light immediately into each vial. The microarray can be read out in parallel with a CCD camera. The experimental setup will be described in section 2.1. Redundancy is built in by performing each test at multiple locations on the microarray during the same screening procedure. The readout of the microarray results in high dimensional data: images combined with data from the other sensors. This enormous amount of data correlated in time and space must be further processed to extract valuable information. The information includes the type of molecules, the concentration of each type, reaction speed, etc. Expert systems will be used to derive new insight in the biochemical processes. The acquired knowledge and expertise will be used to give feedback not only to the present situation in the fermentation tank, but to the entire underlying chemistry. This means that the fermentation process can be optimized, as well as the whole screening methodology.

2. MATERIALS

One speci c project within this interfaculty research program focuses on a sensor system for the acquisition of the

uorescent signals that will be generated in the vials of the microarrays. Our rst approach to develop such a system employs wide eld-of-view imaging, i.e. to image the entire microarray on a CCD camera with low magni cation optics. In this section the experimental setup, the design of the microarrays, and the liquid handling system will be described.

2.1. Detection system

The experimental setup is built around a Zeiss Axioskop epi-illumination microscope system. This microscope is equipped with a fully automated xyz;stage (Ludl Electronics Products Ltd, Hawthorne, NY, USA). A KAF 1400 Photometrics Series 200 CCD camera is mounted on the microscope via a 1:0 camera mount (Diagnostic Instruments Inc., Sterling Heights, MI, USA). The CCD element contains 1317  1035 pixels with a pixel size of 6:8  6:8 m2 . This CCD camera is Peltier cooled to ;42C. Due to this cooling and a slow readout rate (500kHz) this camera is photon limited. The characteristics of this camera in terms of Signal-to-Noise Ratio are excellent.1 This 29

(a) Silicon, circular, wet etched

(b) Silicon, square, wet etched

(c) Silicon, circular, dry etched

(d) Silicon, square, dry etched

(e) Glass, circular, wet etched

(f) Glass, square, wet etched

Figure 1. This gure shows the six dierent types of vials that have been fabricated. The diameter of the circular

masking pattern is 200 m. The width of the square masking pattern is 200 m. The depth varies from 40 m for the vial in Figure 1(a) to 17 m in Figure 1(e) and 1(f).

camera is connected via a NuBus interface to a computer. A Macintosh Quadra 840AV computer takes care of the microscope control and the image acquisition. The microscope as well as the camera can be controlled from within the environment of an image processing package; this package is SCIL Image (TNO Institute of Applied Physics (TPD), Delft, The Netherlands, e-mail address: [email protected]). To image the entire microarray onto the CCD array, a low magni cation objective is used: a 2:5  = 0:075 Zeiss Plan-NEOFLUAR objective. With this objective it is possible to image an area of 3:5  2:7 mm2 onto the CCD array. Using vials with an area of 200  200 m2 and a center-to-center distance of 300 m, i.e. there is an empty region of 100 m between two vials, it is possible to readout 100 vials per image. Wide eld-of-view imaging with low magni cation optics, however, is at the expense of absolute sensitivity. The reason is that the light-gathering power of an objective lens is proportional to the square of the NA: the lower the NA, the less ecient photons can be collected.2

2.2. Design and fabrication of microarrays

Six dierent types of microarrays have been manufactured at DIMES (Delft Institute for Microelectronics and Submicron Technology, Delft University of Technology, NL-2628 CD Delft, the Netherlands). These vials are fabricated either in silicon or in glass. Dierent geometries can be made by using dierent etching mask patterns. For our vials, one mask with circular patterns is used and another mask with square patterns. The dimensions of the patterns are 200 m in diameter or in width respectively. In the following, the fabrication process of the silicon microarrays will be described, followed by the fabrication process of the glass microarrays. The silicon wafers are rst covered with a thin layer of silicon nitride. On top of this layer a thin lm of photo resist is deposited. This lm is exposed through the masking pattern of the microarrays. After removal of the exposed photo resist, the wafer is etched using plasma etching. During this process, the silicon is uncovered at the locations of the vials. The remaining photo resist is removed by rinsing the surface with acetone. Both wet and dry etching techniques were employed on the silicon wafers for the realization of the vials. The wet etching was conducted in potassium hydroxide (KOH). Because of the crystal structure of silicon, this technique results in an anisotropic etching of the silicon. The silicon wafers have a < 100 > surface plane orientation. The etching rate for 30

the < 111 > crystal planes is much slower in KOH solution than for the other crystal planes (typically by a factor of 50). Therefore, wet etching of the silicon results in vials with the shape of a truncated pyramid, which has < 111 > planes as side walls for circular as well as for square etching masks. These two types of vials are shown in Figure 1(a) and 1(b) respectively. In the case of a circular window, underetching of the silicon nitride will take place resulting in free hanging silicon nitride in the corners of the vial, as can be seen in Figure 1(a). The angle between the < 111 > planes and the < 100 > plane is about 54:7. The depth of the vials in Si for the largest sizes is typically about 20 m. The dry etching is conducted by Reactive Ion Etching (RIE), which results in an anisotropic etching. The RIE etched vials have a cylindrical or cubic shape after etching with a circular or square masking pattern respectively. These vials are shown in Figure 1(c) and 1(d). Microarrays have also been manufactured in glass. Glass wafers are covered with a thin layer of polysilicon, which is then patterned to form the etching mask. Wet etching in a hydro uoric acid (HF) solution is utilized. Glass is an amorphous material, and thus isotropic for etching. This process results in a cilindrical or cubic shape with rounded edges and corners. These shapes are shown in Figure 1(e) and 1(f). In Table 1, typical depths and volumes are given for these six shapes of vials. The dierent types of vials are lled according to the methods described in the next section.

Table 1. Typical depths and volumes for the six dierent shapes of fabricated vials with 200 m diameter or width. Material Etching Pattern Depth (m) Volume (nl) Silicon wet etched circular 40  1:6 square 20  0:8 Silicon dry etched circular 23  0:7 square 23  0:8 Glass wet etched circular 17  0:5 square 17  0:7 Each microarray contains 5  5 vials. The center-to-center distance is 600 m. The dimensions of the microarray itself are 2  1 cm2 . In Figure 2 one of the microarrays is shown.

1.0 cm

2.0 cm

5x5 square wet etched vials, 200x200x20 µm3

Figure 2. A microarray with 5  5 vials.

2.3. Fluid injection

One of the diculties involved in the miniaturization of high-throughput screening technologies is liquid handling. Fluid volumes less than a nanoliter need to be injected into small vials. The volumes of the vials described in Subsection 2.2 ranges from  0:5 nl to  1:6 nl. An Eppendorf Transjector 5246 (Eppendorf, Netheler-Hinz GmbH, 22331 Hamburg, GE) is used to inject such small volumes into the vials. This machine is primarily used for In 31

Exponential pressure decay

Sudden pressure build-up

Pressure

Injection Pressure

Injection time

Compensation Pressure Time

Figure 3. The operation of the Transjector: to inject small liquid samples, the Transjector suddenly builds up

a pressure (typically about 150 hPa), which is kept during a certain injection time (typically about 1:5 s). After injection, the presure is exponentially released to the compensation pressure. Vitro Fertilization (IVF), in which sperm cells are injected through the cell membrane into an oval cell. In order to inject sperm cells into an oval cell or in our case to deliver small liquid volumes into a nanoliter vial, a special capillary the Femtotip II is used. The Femtotip II is about 5 cm long and the outlet of the capillary has an inner diameter of less than 0:5 m. The capillary tapers o to the outlet. The problem with this feature is that the ow is not constant towards the outlet of the capillary. Therefore it is not possible to control the injected volume. Other groups have used the same or similar apparatus but with their own pulled capillaries.3{5 These pulled capillaries have a constant diameter. This enables good control of the ow. As a result, the injected volumes with these kind of capillaries are much smaller:  10 ; 100 pl. In order to inject a small liquid sample into a vial, the Transjector builds up a sudden injection pressure. This pressure is held during the injection time. After injection the pressure is exponentially released to the compensation pressure. It is necessary that the tip of the capillary touches the surface of the vial in order to release the liquid drop from the tip and to get the liquid in the vial. The operation of the Transjector is shown in Figure 3. The compensation pressure must always be applied because of the capillary force. Experiments have been done to partially ll the vials, but the variation in the injected volume for a partially lled vial is quite large. Therefore the vials are always lled completely. From the experiments described in Section 4.1 follows that the volume variation of a completely lled vial is about 6%. It is obvious that this kind of liquid handling does not meet our demands. With this device it is only possible to ll a single vial at a time with just one liquid sample. We, however, want to ll an entire microarray at once with many dierent chemicals. Figure 4 shows the experimental setup for lling the vials with a solution.

2.4. Chemical solutions and solvents

For the experiments we have used several dierent solutions of rhodamine and resoru n. In the previous section we have shown that the volume of the vials is on the order of one nanoliter. If a droplet of water of this size is pipetted into a vial, it evaporates in less than a few seconds. In order to reduce the evaporation rate we have used solutions of glycerol : water (1:1, v/v) with a viscosity of 8:30 , where 0 is the viscosity of water. With this high viscosity the evaporation process of the liquid is extended to more than 45 minutes. To lower the surface tension a detergent is added to the solutions. The solutions of Rhodamine B (Merck, Ge) were prepared by serial dilution in a glycerol : water (1:1, v/v) mixture containing 0.005% (w/v) Sarkosyl NL-97 detergent (Cyba-Geigy). The solutions of Resoru n were prepared also by serial dilution in a glycerol : 50 mM sodium phosphate buer, pH 7.4 (1:1, v/v) mixture containing 0.005% (w/v) Sarkosyl NL-97 detergent (Cyba-Geigy). The Sodium phosphate buer and resoru n (sodium salt) were purchased as parts of the Amplex Red Glucose Assay kit (A-12210, Molecular Probes, Eugene, OR, USA). Millipore demineralized water and glycerol (analytical grade reagent, BDH, England) were used for all experiments.

32

3. THEORETICAL ASPECTS OF CONCENTRATION MEASUREMENTS

In this section we will brie y describe some aspects related to our experimental setup for measuring concentrations of uorescing particles in solution. It is possible to divide these aspects into two parts: on one hand the eciency of exciting the uorophores, and on the other hand the eciency of collecting and detecting the emission light from the uorophores.

3.1. Excitation eciency

The strength of the emission light depends on two properties of the uorophore: the quantum eciency or quantum yield and the absorption cross section of the uorophore. The latter property is comparable to the molar extinction coecient or the absorptivity in a bulk solution. The absorption cross section is the eective area of a uorophore which is illuminated by an incident beam of light and which is capable of absorbing photons. The quantum eciency of a uorochrome is de ned as the ratio between the number of emitted photons to the number of absorbed photons.2 The quantum yield of a uorophore must be high, at least up to 30%.6 Furthermore, the excitation eciency is aected by the illumination of the excitation light beam. In Kohler illumination the light beam emerging from the light source is focused in the back focal plane of the objective. This results in a parallel bundle of light rays in the image plane of the objective. As a result the eld-of-view is evenly illuminated. The area of the eld of view is proportional to 1=M2, where M is the magni cation of the objective. As a result, the light intensity per unit area in the eld-of-view is proportional to M2 . With wide eld-of-view imaging, the total energy of the incoming light is spread out over a large region, where the light is not wanted. Given a microarray with vials of sizes 200  200 m2 and a 600 m center-to-center distance, the area of interest is just 10%. In the remaining 90% of the eld-of-view re ections from the surface of the microarray occur. These re ections cause a high background signal. A second source of in uence is stray light which originates in the optical system by re ections ( are) at air-lens interfaces and scattering at diuse surfaces within the optical system (glare). Finally, the third source of in uence is (Raman) scattering in the solvent and auto uorescence in the objective. These phenomena are mentioned here, because they originate in the excitation path of the microscope system, although they in uence the detection of the uorescence signal.

3.2. Detection eciency

The detection eciency is mostly in uenced by the Numerical Aperture of the objective. The light gathering power of an objective in Kohler illumination is proportional to NA2 =M2 . Under the assumption that there is a linear relation between excitation and emission, we can say that the intensity of the emission light is proportional to M2 , as shown in the previous section. Combining these results, it follows that the light gathering power of an objective (with epi-illumination adjusted to Kohler illumination) is proportional to NA2 . For our experiments we have used a 2:5  = 0:075 Zeiss Plan-NEOFLUAR objective. The cone of light that can be collected with this objective is

Figure 4. The microscope setup for lling the microarrays. The capillary is placed under the 2:5 objective (working distance 9:3 mm) at an angle. The 5  5 200  200 m2 microarray is clearly visible by the scattered excitation light. At the right a "Dutch Dime" (15 mm in diameter) is placed to indicate the size. 33

Coefficient-of-variation (%)

Coefficient-of-variation (%)

8 7 6 5 4 3 2 1

8 7 6 5 4 3 2 1

1

2

3

4

5

6

7 8 9 10 11 12 13 Number of filling procedure

(a) Inter-vial error: 5:9%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Label of vial

(b) Intra-vial error: 5:9%

Figure 5. The left graph shows the inter-vial error for successive lling procedures. This error is expressed as the

coecient-of-variation. On average this error is 5:9%. The right graph shows the intra-well error for dierent vials on the microarray. This error is equal to the inter-vial error. less than one percent of the total solid angle of 4. Under the assumption that the emitted photons radiate in all directions, the light collection eciency of such a low NA objective is less than one percent. The photons collected by the objective fall onto the CCD array and are converted to photoelectrons. This conversion factor is the quantum eciency of the camera and depends on the wavelength of the light. In the UV range of the spectrum the quantum eciency is virtually zero, whereas the quantum eciency towards the red part of the spectrum goes up to about 40 ; 50 % for this CCD camera. Another important characteristic of the CCD camera is the electronic gain factor : the electronic gain for the KAF 1400 Photometrics CCD camera is 0:126 #ADU=e; , i.e. an increase of the intensity with a single grayvalue means that about 8 photoelectrons have been collected.1 The lowest measurable signal with our system is limited by the readout noise of the camera: the statistical distribution of the detection limit should lie well above the distribution of the readout noise in order to discriminate between noise and signal. The readout noise is additive and has a Gaussian distribution. It is therefore expressed in terms of its standard deviation in number of electrons. Due to the slow readout rate of this CCD camera the readout noise is as low as 11.7 electrons.1

4. EXPERIMENTAL SECTION

In this section, we will describe a number of experiments that have been performed with the microarrays. All experiments described below are performed with the vials with the shape of a truncated pyramid (See Figure 1(b)). These vials have dimensions of 200  200  20 m3 . This corresponds to a volume of  0:8 nl. Section 4.1 describes the experiment to measure the variation of the manual lling procedure of the vials. Section 4.2 describes the measurement of the detection limit of our system. In Section 4.3 we will show the in uence of certain modi cations to the system. Finally, in Section 4.4 we will show some aspects of evaporation processes that occurred after lling the vial with a solvent with (too) low viscosity.

4.1. Calibration of the injected volume

As described in Section 2.3 the various solutions are manually injected into the vials with a special capillary. In this experiment we want to calibrate the injected volume in terms of a lling error. The lling procedure is quite tedious. The lighting conditions are bad and the microarrays must be moved under the capillary to ll vial after vial. For this reason this experiment is only performed for one particular shape of vial. The vials used in this experiment have the shape of a truncated pyramid with dimensions 200  200  20 m3 (See Figure 1(b)). We expect that the geometry of the vial has some in uence in the error, however, we assume that this contribution to the error is small with respect to the contribution of the method of lling the vials. In order to measure the lling error, 4  4 vials (corresponding to an area of 2  2 mm2 ) were lled with the same Resoru n solution (1:0 M resoru n in glycerol/water). From each vial the average intensity was measured. The measured intensity is directly proportional to the number of molecules 34

Average vial intensity (#ADUs)

1000 500

100 50 blank 10

10-8

10-6 10-4 Rhodamine concentration (M)

Figure 6. A typical response of the detection system to dierent concentrations of rhodamine. For all six vials the detection limit is on the order of 10;7 ; 10;6 M. in the vial, which is directly related to the amount of liquid in the vial. From these values a lling error can be computed: this is the lling error that occurs after lling a number of vials on the same microarray. Part of this error is caused by the lling method and part of this error is caused by dierences in volume of the vials. The variation in volume of the vials is expected to be quite small. This error is the inter -vial error. This experiment is repeated with the exact same microarray more than ten times, which results in a better estimate of the inter-vial error. From these results, a second error can be de ned: the intra -vial error. Only the lling procedure itself contributes to this error. In Figure 5 the results of this experiment are shown. Both error measures are expressed in terms of the coecient-of-variation, which is de ned as =  100%. As can be seen in Figure 5, the inter-vial error is equal to the intra-vial error 5:9%, which corresponds to about 50 pl. These results imply that the variation of the volume of the vials is negligible with respect to the error of the lling procedure. Although the method to ll the vials is quite tedious, the error is acceptably small.

4.2. Measurement of detection limit

The detection limit for a similar kind of setup with high Numerical Aperture objectives and laser induced uorescence is on the order of 10;9 M.7 We have measured the detection limit of our system by lling the vials with solutions with varying concentrations of rhodamine and measuring the signals from the vials. This experiment is performed for the six dierent vials as shown in Figure 1. A typical graph of this experiment is shown in Figure 6. This graph corresponds to the experiment with the vials of the type as shown in Figure 1(b). This experiment is repeated with resoru n in the same type of microarrays. In this experiment the concentrations of resoru n were restricted from 1:0 to 20:0 M. In Figure 7 an image of the lled microarray with resoru n is shown and the response of the system to the dierent concentrations.

4.3. In uence of modi cations to the system

In this section we will describe some preliminary results of experiments that have been performed to measure the in uence of certain modi cations to the microscope system. With our microscope system it is easy to adjust the illumination to Kohler illumination or to critical illumination. Futhermore, the eld stop can be adjusted to change the eld of view. Finally, objectives with higher Numerical Apertures could have been used to measure its in uence. For the latter modi cation, we do not have any results as yet. The results of the rst two modi cations will be shown below.

 Kohler vs. Critical illumination. In Kohler illumination the image of the arc lamp is focused in the back focal

plane of the objective. This results in a parallel bundle of light in the specimen plane of the objective. This type of illumination leads to a uniformly illuminated eld-of-view. When the illumination system, however, is adjusted to critical illumination, the image of the arc lamp is imaged in the specimen plane. In this case the eld-of-view is very brightly illuminated in the center, but the intensity drops o rapidly towards the border of the eld-of-view. It is obvious that Kohler illumination is required to readout the vials of the microarray in parallel. To measure the in uence of the illumination adjustment we lled vials with dierent concentrations 35

Average vial intensity [#ADUs]

1000 800 600 400 200

5

(a) Image of microarray

10 15 20 Concentration resorufin [µMolar]

(b) System response to resoru n

Figure 7. The image on the left side shows a microarray lled with solutions of varying concentrations of resoru n: each column contains one speci c concentration. The data of this image is shown in the right graph. This graph shows the averaged vial intensity for dierent concentrations of resoru n 2:5 ; 20 M.

Critical illumination (#ADUs)

of rhodamine and measured the uorescence signals as a function of the illumination type. The results of this experient are shown in Figure 8. It can be seen in this graph that with critical illumination 5 times more light comes out of the vial than with Kohler illumination. This does not necessarily mean that the detection limit of the system will move towards lower concentrations. 2000 1000 500 200 100 50 20 20

50

100 200 500 Kohler illumination (#ADUs)

Figure 8. Critical illumination vs. Kohler illumination: With critical illumination 5 more light comes out of the vials than with Kohler illumination. The dierent grayvalues of the dots correspond to dierent concentrations.  The eect of closing the eld stop. In Section 3 we mentioned that with the current microarrays 90% of the

total area is illuminated from which no valuable signals are acquired. This extraneous illumination causes stray light in the optical system. One way to reduce the amount of stray light is closing the eld stop in the excitation light path. In this experiment we measured the uorescent signal from the vials and from the background, when the size of the eld-of-view was adjusted by opening or closing the eld stop. In Figure 9 the results of this experiment are shown. It follows from these results that stray light can be avoided to a certain extent by closing the eld stop. 36

Average intensity Vial (#ADUs)

Background 35 30 Vial

25 20 15 10 5 closed

open intermediate filled Opening of the Field Stop

Figure 9. Closing the eld aperture reduces the amount of stray light in the optical system. Note that the signal from the vial is lower than the signal from the background, which is the empty area of the microarray. Closed is the smallest opening, open is the widest opening, lled means that the edge of the eld stop lls the aperture of the eyepiece. The intermediate opening is between the closed and lled opening.

4.4. Evaporation of solvents

A droplet of water of 1 nl injected into one of the vials evaporates within a few seconds because of its low viscosity. In order to prevent evaporation or at least to extend this process suciently, we have used solvents with a higher viscosity. As described in Section 2.4 glycerol/water (1:1,v/v) with a viscosity of 8:3 times the viscosity of water is used as solvent. Mixtures of ethyleneglycol and water in dierent ratios have a viscosity of about 50 . Once a vial is lled with this solvent, it evaporates in less than 20 minutes. We have monitored this process by acquiring an image every 30 seconds. Five images of this process are shown in Figure 10.

(a) t = 0 min:

(b) t = 5 min:

(c) t = 10 min:

(d) t = 15 min:

(e) t = 20 min:

Figure 10. A series of images showing the evaporation process of ethyleneglycol-water (90%=10%; v=v) in a vial at dierent times.

At t = 0, just after injection of the liquid, the signal from the vial is uniform, besides some geometrical eects along the sidewalls and in the corners of the vial. This implies that the meniscus of the liquid is virtually at and the vial is completely lled. After lling the vial, the liquid is "pinned" to the edge of the vial. During evaporation, the pinning of the liquid ensures that the evaporation from the edge is replenished by liquid from the center part of the liquid in the vial.8 This can be seen qualitatively in Figure 10: the uorescent signal gets weaker in the center, until the bottom of the vial is reached. The signal disappears from that area sometime between 10 and 15 min. While the evaporation continues, the liquid is stuck in the four corners of the vial. At these spots the concentration of uorescing particles is increasing. Finally, when the solvent is completely evaporated, the particles loose their

uorescent behavior and the signal disappears. In Figure 11 the average uorescence signal from the vial and the signals from the center, from one of the sidewalls and from one of the corners of the vial is shown as a function of time. It can be seen in this graph that the average uorescence signal from the vial remains almost constant during 37

intensity in #ADUs

800

600

Intensity in corner

400

Average vial intensity

200

Intensity at sidewall

Intensity in centre 5

10

15

20 time in minutes

Figure 11. The average uorescence signal from the vial, the signals from the center of the vial, the sidewalls of the vial and the corner of the vial as a function of time. It can be seen that, besides some geometrical eects, the average signal of the vial does not decrease during the evaporation process. evaporation. The slight increase of the signal in the beginning is caused by geometrical eects. These eects are less than 10% of the signal. Furthermore, it can be seen that the signal in the corners of the vial increases steadily, because of the accumulation of the uorescent particles at these spots. Note that with our microscope system the three dimensional liquid-air interface, the meniscus, is projected onto a plane. Therefore it is not possible to retrieve the shape of the meniscus during the evaporation. It is possible to monitor this with a confocal scanning laser microscope.

5. CONCLUSIONS AND DISCUSSION

In this phase of our research program, we use miniaturized micro titre plates fabricated in silicon. In the future, these microarrays will have integrated intelligence by means of several sensors and actuators. The volume of each vial is on the order of one nanoliter. In order to pipette such a small volume, a special apparatus is used. With this machine it is possible to manually ll the microarrays. To avoid evaporation, solutions with a high viscosity must be used. We have used glycerol/water as solvent with a viscosity of 8:30 . To lower the surface tension a detergent is added to the solutions. The lling error is 5:9%, which corresponds to about 50 pl. In this phase we have aimed at wide eld-of-view imaging at the expense of absolute sensitivity. We have therefore used an epi-illumination microscope equipped with low magni cation optics and a scienti c CCD camera. With this setup the detection limit is on the order of micromolar concentrations. The system can be improved by reducing the amount of stray light. There are several options to solve this. One option is to separate the excitation path from the emission path in the optical system. A second option is to insert a grating right after the light source. This grating will only pass the light that reaches the area of the vials. A third option is to integrate the light source into the vials. This can be achieved by means of optical waveguides or with light-emitting polymers. At this moment these polymers are only used in displays of portable computers.

ACKNOWLEDGMENTS

This work is supported by the Delft Interfaculty Research Center Intelligent Molecular Diagnostic Systems (DIOCIMDS).

REFERENCES

1. J. C. Mullikin, L. J. van Vliet, H. Netten, F. Boddeke, G. van der Feltz, and I. Young, \Methods for ccd camera characterization," in Proceedings of the SPIE Image Acquisition and Scienti c Imaging Systems San Jose, vol. 2173, pp. 73{84, 1994. 2. F. Rost, Fluorescence Microscopy, Vol. I, Cambridge University Press, Cambridge, Great Britain, 1992. 38

3. R. A. Clark, P. B. Hietpas, and A. G. Ewing, \Electrochemical analysis in picoliter microvials," Anal. Chem. 69, pp. 259{163, 1997. 4. A. L. Grosvenor, C. L. Crofcheck, K. W. Anderson, D. L. Scott, and S. Daunert, \Calibration of micropipets using the bioluminescent protein aequorin," Anal. Chem. 69, pp. 3115{3118, 1997. 5. C. L. Crofcheck, A. L. Grosvenor, K. W. Anderson, J. K. Lumpp, D. L. Scott, and S. Daunert, \Detecting biomolecules in picoliter vials using aequorin bioluminescent," Anal. Chem. 69, pp. 4768{4772, 1997. 6. D. Taylor, A. Waggoner, R. Murphy, F. Lanni, and R. Birge, Applications of Fluorescence in the Biomedical Sciences, A.R. Liss, Inc., New York, 1986. 7. M. Eigen and R. Rigler, \Sorting single molecules: Application to diagnostics and evolutionary biotechnology," in Proc. Natl. Acad. Sci. USA, vol. 91, pp. 5740{5747, June 1994. 8. R. Deegan, O. Bakajin, T. Dupont, G. Huber, S. Nagel, and T. Witten, \Capillary ow as the cause of ring stains from dried liquid drops," Nature 389, pp. 827{829, 1997.

39