DE20220299U1 - Apparatus to scan a biochip, with a coating of molecules on one side, comprises a number of pumps for different fluids in a complete liquid handling system without contamination - Google Patents

Abstract

Apparatus to scan a biochip (32), with a coating of molecules on one side, comprising a number of pumps (16) for different fluids and a fluid feed channel (20) to carry them to a flow cell (24), for contact with the coated side of the biochip, is new. Apparatus to scan a biochip (32), with a coating of molecules on one side, comprising a number of pumps (16) for different fluids and a fluid feed channel (20) to carry them to a flow cell (24), for contact with the coated side of the biochip, is new. The flow cell has a flat base (22) with two drillings for the fluid inflow (26) and outflow (40). The fluid flows through a hollow zone (30), in contact with the molecules. The side walls of the hollow zone form an acute inner angle and a diametrically opposed inner angle for the fluid outflow. A distorting seal (28) is over the hollow zone base.

Description

ATPT02057 .* ,**. . ', , - I * · · · 12/20/02

Applicant:
ATTO-TEC GmbH Schanzenweg 5 0

57076 Siegen

Biochip reader

Description

In more and more medical and scientific applications, samples are analyzed qualitatively or quantitatively using biochips. Biochips include DNA arrays, RNA arrays, protein arrays, in short, all types of arrays in which different types of molecules are specifically arranged on different spots on a carrier. A microscope slide is often used as a carrier. One side of this carrier is coated with different biomolecules in a regular grid, e.g. by means of immobilization.

If a specific hybridization reaction is detected using a biochip, e.g. a DNA array, the sample is placed on the DNA array. The sample contains, for example, DNA or RNA molecules marked with dyes. Hybridization takes place in a special hybridization chamber at a suitable temperature. Non-hybridized or non-specifically bound DNA or RNA from the sample to be examined is removed by rinsing. Hybridized DNA or RNA molecules are detected in a so-called reader according to their marking.

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A reader is usually a device for optically reading fluorescence or chemiluminescence signals emitted by a biochip.

DE 197 36 641 A1 describes a device for reading biochips (biochip reader) in which many components are integrated, including the provision and control of the supply of liquids and an optical recording of the measured values. However, the supply of the required liquids to the biochip in the known device does not guarantee the greatest possible freedom from contamination by liquids from previous work steps.

Therefore, such and similar devices may become contaminated from previous rinsing steps, which can only be reduced by extensive rinsing.

The object of the invention is to facilitate the execution of tests or assays with biochips.
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This object is achieved by the inventions according to the independent claims. Advantageous further developments of the inventions are characterized in the subclaims.

According to the invention, a device for reading a biochip is proposed. Such devices are also referred to as readers. The biochip is, as usual, coated with molecules on one side.

The device has a plurality of pumps for different fluids. The fluids are usually liquids, in particular solutions that contain the sample or are used to rinse the biochip. A control for the pumps is often integrated into the device. The device also has means for guiding the fluids to a flow

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cell in which the coated side of the biochip comes into contact with the fluids. These means can include, for example, a mixer in which the individual liquids taken from individual storage containers are brought together and from which a single line leads to a chamber.

The device therefore contains a complete system for what is known as liquid handling. It is not a matter of individual devices with the often associated communication problems between the different devices.

The flow cell has at least the following components: a substantially flat bottom with two holes that serve as inlet and outlet for the fluids, and a cavity through which the fluids flow and in which they come into contact with the coated side of the biochip. The cavity is delimited by side walls, the bottom and the biochip.

The side walls of the cavity are arranged in such a way that they form an acute interior angle and a diagonally opposite, second interior angle. The inlet is arranged in the acute interior angle, the outlet in the second interior angle.

The cavity can, for example, take the form of a kite square, as is known from simple flying kites, i.e. a square in which one of the two diagonals is the axis of symmetry. The cavity would then have an acute interior angle at the base of the kite, in which the inlet is located, and an obtuse or less acute interior angle at the head of the kite, in which the outlet is located. A section with parallel walls can also be inserted between the two angles. It is also conceivable that the depression has no symmetry.

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metric axis, but its shape is close to a kite.

The shape of the depression results in a laminar flow of fluids or liquids in the chamber. The newly entering liquid fills the chamber almost completely and displaces the liquid or air that is already present. This means that hardly any residue of the liquids from previous reaction steps remains in the chamber.

The biochip is placed on the cavity, with the side coated with molecules facing the cavity. When the fluids are pumped through the chamber, they come into contact with the coated side of the biochip. In this way, the desired detection reactions or assays can be carried out.

As a rule, the reactions taking place are monitored using optical, or more precisely spectroscopic, methods. It is therefore advantageous to place the flow cell in a sample chamber that can be sealed against light so that the optical signal can be recorded without any disturbing background light.

Typically, optical signals are generated either by chemiluminescence or by fluorescence. Chemiluminescence can be read out, for example, using a CCD camera, which simply detects the intensity of the chemiluminescence. To detect fluorescence, a means is required to couple in an excitation light source, the light of which is directed onto the biochip.

In a particularly simple embodiment of the invention, the cavity in the flow cell is formed by arranging a deformable seal above the bottom

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on which the biochip is placed. The seal has a recess in the shape of the cavity in its middle area. In order to seal the cavity against the escape of fluids, the seal is preferably deformable. It can be both elastically and plastically deformable.

Such a seal can be a consumable item, or it can be removable and can be easily rinsed as a single, isolated part without many corners and edges.

Preferably, the seal is made of silicone, which is inert to most relevant liquids.

It has proven to be beneficial if the soil on the

the side facing the flow cell is hydrophobic. A consistently smooth and hydrophobic surface of the bottom prevents air bases from becoming trapped in the chamber when the liquids are pumped through the flow cell, especially when the first liquid enters the air-filled flow cell.
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Advantageously, the bottom or the entire cavity or sample chamber is thermostatted to achieve higher reproducibility. Furthermore, this offers the possibility of controlling reactions using temperature changes, e.g. as in the PCR reaction.

If you are working with very low concentrations, it is advantageous if the fluids are not pumped through the cavity too quickly, as the reactions to be investigated may not take place in the required number within the long diffusion times required. Instead, the fluids can be pumped alternately and left to rest. This offers the possibility of waiting for the necessary diffusion and reaction times.

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The invention is explained in more detail below using embodiments that are shown schematically in the figures. The same reference numbers in the individual figures refer to the same elements. In detail:

Fig. 1 is a schematic representation of the biochip reader;

Fig. 2 the bottom of the biochip reader; and

Fig. 3 the seal.
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Fig. 1 shows the biochip reader 10. The biochip reader 10 contains storage containers 12. The contents of the storage containers 12 are conveyed via lines 14 by pumps 16 (usually eight in number) to a mixer 18. The mixer 18 is connected to the base 22 of the flow cell 24 via a line 20 (preferably a Teflon hose). The line 20 is connected to the base via a connection piece 26. On the base there is a seal 28 which has a cavity 30. The biochip 32 is placed on the seal 28.

The flow cell 24 is located in a light-tight sample chamber 34, which is closed by walls 36 and a lid 38. The lid 38 has (schematically shown) stamps 39, which press on the biochip 32 and thereby seal the cavity 30 hermetically.

The liquids pumped through the line 20 into the cavity 30 flow through a drain 40 via a line 42 (also preferably a Teflon hose), supported by a further pump 44, into a collecting container 46.

The base 22 is made of transparent material (see below). Below the base 22 there is a camera,

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preferably a CCD camera 48, which collects the light emitted in the cavity 30 or on the biochip 32 via an optics 50.

The biochip reader is controlled by a computer 52, which is connected to the biochip reader 10 via a data connection 54. Inside the biochip reader 10 there are a number of data lines (not shown) that establish the connection between the computer 52 and the individual components of the biochip reader 10.

The flow cell 24 consists of three parts, the base 22 with the connections for the two Teflon tubes 20, 42, the seal 28 and the biochip 32, a coated carrier made of glass. The three parts are placed one on top of the other in the sample chamber 34 in this order.

Pressure exerted by the lid 38 of the sample chamber 34 via the plungers 39 on the biochip 32 forms the sealed cavity 30 through which the liquids, usually solutions, are flushed.

In order to prevent the biochip 32 from bending or even breaking, the surface of the cover 38 of the sample chamber 34 which exerts the contact pressure on the biochip 32, i.e. the stamp 39, is designed in the same shape as the seal 28. As a result, the cover 38 only presses on the biochip 32 where it is supported on its underside by the seal 28. The cover 38 does not press on the biochip 32 where it is freely supported above the base 22.

The volume of the cavity 3 0 is approximately 0.25 ml. With a typical liquid flow of 15 ml / min., the flow velocity under the small, coated portion of the biochip 32 is approximately 40 mm / sec. The coated portion of the biochip 32 typically has a size of 1 cm

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x 1 cm. There the volume is exchanged approximately three times per second; the entire volume of the cavity 30 is renewed within one second.

a) The soil

Fig. 2A shows a bottom view of the base 22. Fig. 2B shows a sectional side view. The base 22 is made of transparent optical quality polycarbonate plastic

(e.g.: Makroion® CD2005). The base 22 typically has a length of 76 mm and a width of 86 mm. The connecting pieces 26, 40 open into through holes 56 through which the fluids or liquids enter the cavity 30.

The plane-parallel surfaces of the base 22 in its central region allow optical observation of the biochip 32 through the base 22.

The upper side of the base 22 is hydrophobic. The consistently smooth and hydrophobic upper side of the base 22 prevents air bases from adhering to the chamber.

b) The seal

Fig. 3 shows the seal 28. The seal consists of a film of soft silicone with a thickness of preferably 0.5 mm. A thickness of 0.25 mm is also possible, which means that the volume of the cavity 30 is halved and the flow speeds are doubled accordingly.

An area is punched out of the film of the seal 28. This creates an acute interior angle 58 of approximately 13° in the right corner of the punched-out area in Fig. 3. In

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In the left corner of the punched-out area in Fig. 3, a second interior angle 60 of approximately 62° is formed.

The punched-out area forms the cavity 30 through which the solutions are pumped. The geometry of the chamber results in a laminar flow profile with an almost straight flow front in the area of the coated part of the biochip. In this way, the surface of the biochip on the carrier is evenly washed.
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c) The biochip

The Biochip 32 is a coated carrier. The carrier can be made of glass or plastic, such as polycarbonate. Typically, a microscope slide is used for the carrier. It is designed as a consumable so that as few cleaning steps as possible are required and no residues of fluids from previous assays remain.

The carrier consists, for example, of glass, the surface of which has been pretreated to bind proteins. Preferably, the surface of the carrier is hydrophobized at the same time. When the chamber is flushed with aqueous solutions, the hydrophobic properties of the surface of the carrier show the same advantages as the hydrophobic surface of the base 22, namely that no air bubbles are held in place. The carrier is typically 76 mm long and 26 mm wide.

d) Detection

If the reaction taking place on the biochip is monitored with a CCD camera 48, a spatial resolution on the

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Biochip 32 of better than 20 μιη can be achieved. Preferably, a cooled CCD camera 48 with 752x528 pixels is used. With a conventional collecting optics 50, slightly more than 0.5% of the photons emitted on the biochip 32 can be captured by the CCD camera.
4 8 can be demonstrated.

The dynamic range of the measurement for one minute is about 10^4. The absolute measuring range for a measurement duration of one minute is from 20 fW / cm ^A 2 to 0.2 nW / cm ^A 2 luminance
on the chip. By adjusting the measurement time, the
absolute measuring range can be changed in both directions.

To ensure that the optical signal can be recorded without disturbing background light, black colored Teflon is used as the material for cables 14, 20 and 42. Lighter colored cables could potentially conduct light.

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ATPT02057 * * ··· ·- 1*L · · · · Reference symbol Biochip reader Storage container cables 10 pump 12 mixer 14 Line 16 Floor 18 Flow cell 20 Connection piece 22 poetry 24 cavity 26 Biochip 28 Rehearsal room 30 Walls of the rehearsal room 34 32 Sample chamber cover 34 34 Rubber stamp 36 Drain 38 Line 39 pump 40 Collection container 42 CCD camera 44 optics 46 calculator 48 Data Connection 50 Through holes 52 acute interior angle 54 second interior angle 56 58 60

Claims (4)

1. Device for reading a biochip ( 32 ) whose one side is coated with molecules, a) with a plurality of pumps ( 16 , 44 ) for different fluids; b) means ( 20 ) for guiding the fluids to a flow cell ( 24 ) in which the coated side of the biochip ( 32 ) comes into contact with the fluids; c) wherein the flow cell ( 24 ) comprises at least the following components: 1. a substantially flat bottom ( 22 ) with two bores ( 56 ) serving as inlet ( 26 ) and outlet ( 40 ) for the fluids; 2. a cavity ( 30 ) through which the fluids flow and in which they come into contact with the coated side of the biochip ( 32 ); 3. wherein the cavity ( 30 ) is delimited by side walls, the bottom ( 22 ) and the biochip ( 32 ); characterized , a) that the side walls of the cavity ( 30 ) are arranged such that they form an acute interior angle ( 58 ) and a diagonally opposite, second interior angle ( 60 ); b) that the inlet ( 26 ) is arranged at an acute interior angle ( 58 ); and c) that the drain ( 40 ) is arranged in the second interior angle ( 60 ). 2. Device according to the preceding claim, characterized in
that the cavity ( 30 ) in the flow cell ( 24 ) is formed thereby,
that a deformable seal ( 28 ) can be arranged above the floor ( 22 ),
that the biochip ( 32 ) can be placed on the seal ( 28 ), and
that the seal ( 28 ) has a recess in the form of the cavity ( 30 ) in its central region. 3. Device according to the preceding claim, characterized in that the seal ( 28 ) is made of silicone. 4. Device according to one of the preceding claims, characterized in that the bottom ( 22 ) is hydrophobic on the side facing the flow cell ( 24 ).