WO2006035392A1

WO2006035392A1 - Biosensors for the analysis of samples

Info

Publication number: WO2006035392A1
Application number: PCT/IB2005/053152
Authority: WO
Inventors: Joost Kahlman; Menno Prins
Original assignee: Koninklijke Philips Electronics N. V.
Priority date: 2004-09-27
Filing date: 2005-09-23
Publication date: 2006-04-06

Abstract

The invention relates to a system (l a) for the examination of a liquid biological sample (2) in a container (21), the system comprising a plurality of individually addressable biosensors (10) distributed in the sample. External transmitters (223) and external receivers (22) are provided that allow to supply power wirelessly and simultaneously to all biosensors (10) while addressing and controlling them individually.

Description

Biosensors for the analysis of samples

The invention relates to a miniaturized biosensor that is adapted for making measurements in a biological sample and for a wireless communication. Moreover, it relates to a system and a method for the analysis of a sample with the help of such biosensors.

The EP 1 252 860 Al discloses a cardiac monitoring system comprising a number of implanted transponders. If a transponder receives external control signals of a suited (radio-) frequency, it sends a reply signal that is modulated according to a measured parameter characterizing the environment of that transponder, for example a temperature. Moreover, the position of the transponder can be derived from its reply in order to derive geometric parameters like heart volume.

The use of transponders on microchips is also known in the area of biochemical analysis. A transponder may for example comprise a specific DNA oligonucleotide probe attached to it and a unique serial number for detecting specific DNA in clinical samples. Specific nucleic acid sequences in biological specimen are found by reacting fluorescein labeled specimen nucleic acid with separate microchips, each carrying different (unique) oligonucleotide probes. A fluorometric scanner then generates laser-light, measures the fluorescent signal generated by hybridized specimen nucleic acid that has attached to the probe on the micro-chip, powers the micro-chip (via photo-voltaic cells) and reads the unique serial number which is transmitted via radiofrequency (RF) by the microchip. The equipment needed for this procedure (e.g. flow cytometer, fluorometric scanner) is however rather expensive and complex.

Based on this situation it is an object of the present invention to provide means for a simple and flexible analysis of- preferably liquid - samples with biosensors. This object is achieved by a biosensor according to claim 1, by a system according to claim 7, and by a method according to claim 9. Preferred embodiments are disclosed in the dependent claims.

According to its first aspect, the invention comprises a miniaturized biosensor, i.e. a sensor for measuring physical, chemical, biological or other parameters that are preferably related to a biological sample. The "miniaturization" of the biosensor means that it has a comparatively small size with a typical diameter ranging from about 10 μm to about 1000 μm. The biosensor comprises the following components:

A receiver for wirelessly receiving external control signals. Said control signals may for example be carried by electromagnetic radiation, preferably by radiofrequency RF (about 10 kHz to about 100 GHz) or visible light.

A powering unit for the generation of electrical power from the aforementioned external control signals. - A sensor unit for measuring (quantitatively and/or qualitatively) at least one parameter of the environment of the biosensor. Said parameter may be, without being limited to, one of the following entities: the binding of a specific molecule, the concentration of a target substance, the pH value, a temperature, a pressure, a motion and/or a field strength (e.g. of an external magnetic field). Examples for special realizations of a sensor unit may be found in the WO 2004/ 53491 Al which is completely incorporated into the present application by reference.

A transmitter for a wireless transmission of data from the biosensor to an appropriate external receiver, wherein said data shall particularly include measured values of the aforementioned parameter. The wireless transmission is preferably based on RF signals.

A control unit for the evaluation of external control signals received by the receiver, wherein said evaluation comprises the detection of an individual addressing of the biosensor and the selective execution of processing steps. With other words, the control unit monitors control signals received by the receiver for the occurrence of the individual address of the biosensor and, in case this address is detected, initiates the execution of processing steps, for example according to commands encoded in the control signals or to a preprogrammed procedure.

An advantage of the biosensor described above is that a plurality of such biosensors can be used for a fast and flexible examination of a (biological) sample because all biosensors can be powered simultaneously by the external control signals and thus can perform their energy consuming measurements permanently. In spite of this simultaneous operation, an individual control and for example readout of each biosensor is possible, too, because they are equipped with individual addresses. The biosensor may at least partially be built as a "chip" of a semiconductor material with methods known from the manufacture of integrated circuits. In this case components like antennas may be integrated on the chip, too, or be provided externally and bonded to the chip.

Due to the simultaneous supply of power to the biosensors and the wireless communication, a plurality of said biosensors may be distributed throughout a (liquid) sample, thus coming into intimate contact with the material to be examined. In order to protect the biosensor during a contact with electrically conducting and/or aggressive materials, the surface of said biosensor is preferably covered with an (electrical, chemical, ...) isolation where necessary. Of course sensitive areas of the sensor unit that must come into contact with the sample will be left out from such an isolation.

According to a further development of the invention, the biosensor comprises means that prevent sticking together of said biosensor with other biosensors of the same kind. Said means may for example comprise protrusions from the surface that prevent a close contact between biosensors and/or repellent surface coatings. As the biosensor is in general not mechanically linked to external control devices, it is preferably equipped with "anchors" for external fields that allow the exertion of forces on the biosensor. These anchors may particularly comprise a magnetic material that may interact with an external magnetic field.

According to another aspect, the invention relates to a system for the analysis of a sample, particularly a biological fluid. The system comprises the following components:

A container for storing a sample during its examination. Said container may in principle have any geometry and consist of any material that are suited for the intended application. In particular the container may be a cylinder or a cuboid of glass or a transparent plastic.

A plurality of biosensors that can be addressed individually in a wireless communication process. The biosensors may particularly be biosensors of the kind described above.

An external transmitter for the transmission of control signals to biosensors that are present in a first region of the container. Said control signals may particularly be carried by electromagnetic radiation, e.g. radiofrequency or light. The external transmitter is preferably disposed outside the aforementioned container, in which case the walls of the container should be transparent for the control signals.

An external receiver for receiving signals from biosensors that are present in a second region of the container, wherein said signals may particularly be carried by electromagnetic radiation (e.g. RF). Moreover, the external receiver is preferably disposed outside the container, and it may share hardware with the external transmitter (e.g. antennas). The aforementioned first region may particularly be identical to the second region, i.e. a bidirectional communication with the biosensors may take place in that region. The system preferably also comprises an external control unit for controlling the operation of the external transmitter and the external receiver and for the evaluation of data received from the biosensors. Such an external control unit may for example be realized by a microcomputer. The system has the advantage that a sample inside the container can be investigated by a plurality of biosensors simultaneously, wherein the position of these biosensors must not be fixed or known for a correct evaluation because the biosensors can be individually addressed. Therefore, the origin of measurement data can unambiguously be associated with the corresponding biosensor.

According to a preferred embodiment of the aforementioned system, the external transmitter and/or the external receiver is arranged in such a way that it can reach biosensors anywhere and at any orientation within the container. In this case the sample may particularly be a liquid with which the biosensors are intimately mixed. Despite of the resulting random and unknown distribution of the biosensors throughout the sample, they can all be controlled and read out correctly by the external transmitter and/or the external receiver.

The invention further relates to a method for the analysis of a sample in a container, said method comprising the following steps:

Filling the sample in a container.

Distributing a plurality of biosensors, which can be individually addressed and which are adapted for a wireless communication, within the sample. Said biosensors may particularly be biosensors of the kind described above.

Making at least one measurement with said biosensors. Addressing at least one of the biosensors by a wireless control signal and wirelessly reading out the measuring data of said biosensor.

The method comprises in general form the steps that can be executed with a system of the kind described above. Therefore, reference is made to the preceding description for more information on the details, advantages and improvements of that method.

According to a preferred embodiment of the method, the biosensors are distributed over a larger volume of a liquid sample during at least a part of their measurements. Subsequently, the biosensors are concentrated or collected in a sub- region of the container for the wireless readout of their measurements. This method has the advantage that measurements of the biosensors can be made within the whole volume of the sample, while the readout process is limited to a sub-region which is more readily accessible to (smaller) external transmitters and receivers. These and other aspects of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter.

In the following the invention is described by way of example with the help of the accompanying drawings in which: Fig. 1 shows a principal sketch of a system for the analysis of a sample fluid according to a first embodiment of the present invention wherein the biosensors may freely float within the sample;

Fig. 2 shows a principal sketch of a system for the analysis of a sample fluid according to a second embodiment of the present invention wherein the biosensors are localized;

Fig. 3 depicts the circuit diagrams of a biosensor and the corresponding external control device with a RF/RF coupling;

Fig. 4 depicts an alternative embodiment of the circuits of Figure 3 with a RF transmitter on the biosensor;

Fig. 5 depicts alternative circuit diagrams of a biosensor and the corresponding external control device with an optical/RF communication.

Figure 1 shows a first embodiment of a system Ia according to the present invention which is used for the analysis of a biological sample fluid 2 in a container 21. Said container 21 may for example be a cylindrical or cuboid glass vessel. Within the sample 2, a plurality of (for example 10 to 10⁷) "free-swimming" miniaturized biosensors 210 is distributed randomly. Said biosensors 210, which will be described in more detail below, are adapted to make parameter measurements inside the sample 2, for example to detect the presence of a target DNA strand or of other bio- molecules (electrolytes, metabolites, proteins, hormones, drugs, pharmaceutical materials, cell fragments, tissue elements etc.), wherein each biosensor can be dedicated to the measurement of another parameter or the same parameter. The biosensors 210 can also be used for measuring biological process parameters like temperature, pH, or flow (e.g. by counting the number of chips per unit of time passing a reference line). Moreover, the biosensors 210 are adapted to communicate wirelessly with their environment, wherein each biosensor 210 can individually be addressed and selectively send its measurement data. The biosensors 210 preferably extract their energy of operation from the control signals 231 with which they are controlled. Typical dimensions of the biosensors are an area of about 0.4 mm² with a thickness from about 10 to 60 μm. In the embodiment shown in Figure 1, external transmitters 223 are arranged around the container 21 in order to constitute a three-dimensional light source for the emission of optical control signals 231 that reach the biosensors 210 inside the sample 2 irrespective of their location and orientation. The optical control signals 231 of the transmitters 223 provide both power to the biosensors 210 as well as control commands. Moreover, RP coils 22 are disposed around the container 21 as external receiver which can receive radiofrequency signals sent by the biosensors 210 irrespective of their position and orientation.

Preferably measures are taken to avoid sticking of the biosensors 210 to each other and to the wall of the container 21 , e.g. by applying roughness to their surface (providing structures to the surface with physical structures, e.g. needles, in order to reduce the contact area), or with repellent surface chemistries.

Another approach to avoid sticking of the biosensors and to guarantee continuous power and data transfer is the immobilization of the biosensors on a surface while transferring the sample fluids across them. A corresponding system Ib is shown in Figure 2. The system Ib comprises a container 121, for example a flat tube, through which a liquid sample 2 flows (arrow). A plurality of individually addressable biosensors 10 is fixed to the wall of the container 2 such that each biosensor 10 has a fixed place. The advantage of this setup is that the communication with the biosensors 10 must not be possible everywhere within the whole sample but only within a limited region at its wall. Therefore, a RF coil 22 at the outer side of the container wall suffices for communication an power supply. Compared to devices known in the state of the art, the system 1 b of Figure 2 is far simpler because no spatial resolution and no direct electrical wiring is required for readout due to the fact that each chip is addressable independently.

According to a further development, the two methods associated with the systems of Figures 1 and 2 can be combined: After the bio-reaction performed by free- swimming biosensors 10, the biosensors are immobilized on the surface of the container and read out. This measure eases readout. In more detail, a preferred assay may comprise the following steps:

Insert a sample (e.g. plasma), reagents (e.g. heparin, labeled bio- molecules) and biosensor chips into a vessel (e.g. well of a microtiter plate). Incubate (i.e. let react and bind), while stirring the solution. Meanwhile detect signals to monitor the binding processes.

Attract biosensors to the wall (e.g. by magnetic forces, by actuated flow, or by letting them settle in gravity) and perform a washing step. Meanwhile detect to monitor the washing process.

Analyze the signals with a dedicated algorithm. Stirring can be done mechanically, but also magnetically when the biosensors are provided with a magnetic layer (e.g. on the back side).

In both cases of Figures 1 and 2 (in- fluid as well as fixed to a body), the system is modular. The test is composed of several different chips. This modular system approach allows custom-specific assembly of a sensor system. The system can be used for diagnostics (e.g. clinical, food, environment, veterinary) as well as for research and development purposes (e.g. high-throughput screening, medical, pharmaceutical, life sciences). Figure 3 shows in more detail a first embodiment of a biosensor 10 and a corresponding external base station 24. The biosensor 10 comprises the following components:

A receiver comprising a resonance circuit with an antenna 11 for the reception of RF signals 31 and for the transmission of signals 32. - A powering unit 13 that is coupled to the receiver and that collects and stores electrical energy from received radiofrequency signals 31.

A control unit or demodulator 12 coupled to the receiver for monitoring the received control signals 31 in order to detect if the particular biosensor 10 has been addressed by the external control signals 31. If such an addressing is detected, the demodulator initiates preprogrammed and/or commanded steps, for example the transmission of a signal 32 via the RF modulator 14 and the antenna 11, said signal 32 encoding data measured by the sensor unit 15.

The already mentioned sensor unit 15 which performs the desired measurement of a parameter.

Figure 3 also shows the external base station 24 which comprises an antenna coil 23 for the transmission of RF control signals 31 and the reception of data signals 32 from the biosensor 10.

The biosensor chips 10 of Figure 3 are powered, controlled and read-out by using RF/RF coupling, a technique known from wireless smart cards and tagging devices. This method has the advantage that the signals 31, 32 are not hindered even when the liquid is optically scattering or non-transparent. Power and data towards the chip is sent via a modulated RF source (RF TX) in the base station. The chip 10 sends data back by field modulation, in fact by "shorting" the RF magnetic field from the RF source. By measuring the voltage (RF RX) on the antenna coil 23 the chip data can be detected. The base station preferably enables one single on-chip RF transmitter each time in order to avoid collision between the RF signals from the chips.

Figure 4 depicts an alternative embodiment of a biosensor 110 and a base station 124, wherein like components as in Figure 3 carry the same reference numbers plus 100 and need not to be explained again. The difference with respect to Figure 3 is that the biosensor 110 now comprises a RF transmitter 114 coupled to a RF antenna 116 for the active emission of RF signals 132 that may particularly encode the data measured by the biosensor 110. Moreover, the external base station 124 now comprises a separate receiver 125 coupled to an antenna 126 for the reception of the signals 132. Alternatively, the reception might also be realized as in Figure 3 via the coil 123.

Figure 5 depicts another alternative embodiment of a biosensor 210 and a base station 224, wherein like components as in Figure 3 carry the same reference numbers plus 200. The difference of this embodiment to that of Figure 3 is that the control signals 231, which transport both power and address information to the biosensor 210, are now carried by visible light. Said light signals 231 are emitted by a light source 223 that is modulated by a light modulator 226. The reception of RF signals 232 from the biosensor 210 is accomplished by an RF receiver 227 with an antenna. Both the light modulator 226 and the RF receiver 227 are coupled to and controlled by a control unit 225.

The biosensor 210 comprises a photo voltaic cell 217 for transforming the light 231 from the modulated light source 223 into power for the chip 210 and for detecting commands from the modulated light source in the base-station 224.

Furthermore an on-chip RF transmitter 214 is present for sending a measured value indicative to the status of the bioassay to the base station 224. The control unit or demodulator 212 extracts control commands from the modulated light source 223 for controlling the biological assay 215 and the on-chip RF transmitter 214. The bio-assay on the chip can be based on a redox reaction, on the detection of magnetic particles, impedance, temperature, etc. It is a fundamental feature of the proposed systems that each biosensor chip 10, 210 has its own unique address so that the base station 24, 224 can control and communicate with each chip individually. Many variations are imaginable for keeping track of the relation between the "logical" chip addresses and the biomaterial on the chip. The addresses can for example be programmed during chip manufacturing or during spotting (e.g. ink-jet printing) of the biomaterial on the chip. In all cases the relation between the logical chip address number and the biomaterial must be kept unambiguous by proper bookkeeping in the base stations or by programming a code indicative to the biomaterial into the chip during spotting of the biomaterial. Prior to the bio-measurement the base station can "scan" all the chips present in the assay. The data rate of the wireless interface between base stations 24, 224 and biosensors 10, 210 is determined by the available bandwidth and the signal to noise ratio of the communication channel. These two variables are largely affected by the regulation rules (transmitting power, bandwidth) of the government. Typical communication speeds are 100 kbit/s, as e.g. used in wireless tags operating in the 13.56 MHz ISM band. When assuming that 100 bits have to be communicated each measurement, 1000 measurement points can be read successively every second. The transmitting power and the bandwidth can be raised up for a biosensor application when the communication takes place in a RF shielded box. Alternatively data comprising the results of the total bio-measurement is stored into the memory of the biosensor chip and read out after the measurement has taken place. This method saves communication overhead and is thus more power/bandwidth effective.

Because of the conducting properties of the surrounding liquid 2, a coating is preferably applied which protects the saw-edges of the biosensor chips. In addition the sensitive area of the chip is protected in such a way that the liquid cannot disturb the functionality.

While the Figures 3-5 suggest an integration of the antennas 1 1, 111, 116, 216 on the chip, it is also possible to use external antennas for this purpose which are bonded to the chip with the residual circuits, wherein the whole biosensor- arrangement is for example located on a common substrate.

At relatively low frequencies, magnetic coupling is also an option for the communication of data and power between biosensor chips and external control devices. In this case, a relatively large antenna is connected to the chip in order to comprise enough magnetic flux.

In RF power transmission where the area/volume power density is not so high, a substantial coil-area on the chip is required to gather enough power. In optical power transfer, the chip area can be smaller because the light can easy be concentrated on a small chip area. The overall power efficiency of the modular biosensor system depends on the power coupling efficiency and on the efficiency of the power converters itself (RF transmitter, antenna, light source, chip-coil, Photo Voltaic Cell etc.).

In summary, the following advantages are associated with a system of the kind described above: - Single chip solution, no external sensors, like a fluorescent microscope, needed for reading the result of the biochemical reaction. Easy multiplexing of multiple bio tests in a single liquid. Wireless readout.

Easy applicable by using standard technology. Finally it is pointed out that in the present application the term

"comprising" does not exclude other elements or steps, that "a" or "an" does not exclude a plurality, and that a single processor or other unit may fulfill the functions of several means. The invention resides in each and every novel characteristic feature and each and every combination of characteristic features. Moreover, reference signs in the claims shall not be construed as limiting their scope.

Claims

CLAIMS:

1. Miniaturized biosensor (10, 110, 210), comprising:

- a receiver (11, 11 1, 217) for wirelessly receiving external control signals (31, 131, 231);

- a powering unit (11, 13, 111, 113, 213, 217) for the generation of electric power from said external control signals (31, 131, 231);

- a sensor unit (15, 115, 215) for measuring at least one parameter of the environment;

- a transmitter (1 1, 116, 216) for a wireless transmission of data (32, 132, 232) including measured values of said parameter;

- a control unit (12, 112, 212) for the evaluation of external control signals (31, 131, 231), said evaluation comprising the detection of an individual addressing of the biosensor (10, 110, 210) and the selective execution of processing steps.

2. The biosensor (10, 1 10, 210) according to claim 1, characterized in that the receiver (11, 111) is adapted to receive control signals carried by electromagnetic radiation, particularly by radiofrequency (31) or light (231).

3. The biosensor (10, 110, 210) according to claim 1 , characterized in that the sensor unit (15, 115, 215) is adapted to measure the binding of specific molecules, a concentration of a target substance, a temperature, a pressure, a pH value, motion, and/or field strengths.

4. The biosensor (10, 1 10, 210) according to claim 1, characterized in that its surface is at least partially covered with an isolation.

5. The biosensor according to claim 1, characterized in that it comprises means that prevent sticking together with other biosensors of the same kind.

6. The biosensor according to claim 1 , characterized in that it is provided with a magnet for the exertion of external magnetic forces on the biosensor.

7. A system (Ia, Ib) for the analysis of a sample (2), comprising

- a container (21, 121) for storing said sample (2) during its examination;

- a plurality of biosensors (10, 110, 210) that can be addressed individually in a wireless communication process, particularly of biosensors (10, 110, 210) according to one of claims 1-6;

- an external transmitter (23, 123, 223) for the transmission of control signals (31, 131, 231) to biosensors (10, 110, 210) that are present in a first region of the container (21, 121); - an external receiver (22, 23, 123, 227) for receiving signals (32, 132, 232) from biosensors (10, 110, 210) that are present in a second region of the container (21, 121).

8. The system (Ia, Ib) according to claim 7, characterized in that the external transmitter (23, 123, 223) and/or the external receiver (22, 23, 123, 227) is arranged in such a way that it can reach biosensors (10, 110, 210) anywhere and at any orientation within the container (21).

9. A method for the analysis of a sample (2), comprising the following steps: - filling the sample (2) into a container (21, 121);

- distributing a plurality of biosensors (10, 1 10, 210) in the sample which are individually addressable and adapted for a wireless communication, particularly of biosensors (10, 110, 210) according to one of claims 1-6;

- making at least one measurement with said biosensors (10, 110, 210); - addressing at least one of the biosensors (10, 1 10, 210) by a wireless control signal (31, 131, 231) and reading out measured data (32, 132, 232) from said biosensor wirelessly.

10. The method according to claim 9, characterized in that the biosensors (10, 110, 210) are distributed over a volume of a liquid sample (2) while making at least a part of their measurements, and that the biosensors (10, 110, 210) are subsequently concentrated in a sub-region of the container (21) for the readout procedure.