Abstract:
The invention relates to a method and a corresponding arrangement for sequencing at least two biopolymers ( 6 ), wherein for each biopolymer ( 6 ) a sequence signal is picked up by a respective measured variable pickup on the basis of the sequence of the biopolymer ( 6 ), the sequence signals are transferred to a shift register ( 16 ) and buffer-stored therein, the buffer-stored sequence signals are transferred from the shift register ( 16 ) sequentially to an evaluation device ( 26 ) and evaluated therein. Each sequence signal is preferably produced here by means of a nanopore arrangement ( 10 ). A corresponding sequencing arrangement ( 11 ) has the measured variable pickups and the shift register ( 16 ) integrated in it, preferably in an electrical circuit, that is to say on a sensor array, for example. Each sequence signal can be amplified here by a preamplifier ( 14 ) prior to transfer to the shift register ( 16 ). The transfer of the output signal (A) to the evaluation device ( 24 ) can comprise the amplification of the signal by an output amplifier ( 24 ) and/or at least one EMCCD stage ( 32 ).

Description:
[0001]    This application is the National Stage of International Application No. PCT/EP2014/059468, filed May 8, 2014, which claims the benefit of German Patent Application No. DE 10 2013 215 666.9, filed Aug. 8, 2013. The entire contents of these documents are hereby incorporated herein by reference. 
     
    
     BACKGROUND 
       [0002]    The present embodiments relate to sequencing at least two biopolymers. 
         [0003]    When sequencing a biopolymer (e.g., a nucleic acid or a protein), the sequence of the individual building blocks of the biopolymer (e.g., the nucleotide sequence (or base sequence) or the amino acid sequence) may be analyzed. 
         [0004]    During sequencing (e.g., with the aid of nanopores), the biopolymer passes through a biological or artificial nanopore. Individual building blocks of the biopolymer may be analyzed by the nanopore as a result of a change in the pore resistance as the building blocks pass through. When different building blocks pass through the nanopore, a current conducted through the nanopore changes, for example. This change is dependent on the building block that passes through the pore, such that, for example, an individual nucleotide may be detected and, for example, the sequence of the nucleic acid may be determined. 
         [0005]    Alternatively, a tunneling current may be measured in the nanopore, the tunneling current occurring only when the biopolymer passes through. The current intensity of the tunneling current is dependent on, for example, the nucleotide or the amino acid that is situated in the nanopore. 
         [0006]    The difficulty, for example, when measuring the tunneling current results from the low intensity thereof in the nanoamperes range and also the relatively high capacitance of the tunnel electrodes. The capacitance arises as a result of parasitic capacitances with respect to the silicon of a silicon wafer on which the nanopore structure may be arranged, and with respect to the surrounding liquid that is in direct contact with the electrodes. Consequently, measuring the tunneling current necessitates preamplifiers that have a current input withstand a relatively high input capacitance (M. Carminati, G. Ferrari, M. Sampietro, A. P. Ivanov, T. Albrecht: “Low-Noise Dual-Channel Current Amplifier for DNA Sensing with Solid-State Nanopores,” 19th IEEE International Conference Electronics, Circuits and Systems (ICECS) 2012, 817-820). 
         [0007]    The article by Carminati et al. describes suitable discrete circuits for measurement on individual micropores. The publication by Rosenstein et al. (J. K. Rosenstein, M. Wanunu, C. A. Merchant, M. Drndic, K. Shepard: “Integrated nanopore sensing platform with sub-microsecond temporal resolution,” Nature methods, 9, 2012, 487-492) describes the use of a complementary metal-oxide semiconductor (CMOS) sensor for detecting a tunneling current. 
         [0008]    With the methods described in the prior art, the throughput of sequencings of a plurality of samples is not as high as desired, however. Sequence signals (e.g., signals detected by a measurement variable pick-up depending on a detected building block of a biopolymer) have a low current intensity and a small quantity of charge and are often superposed by noise. This problem also arises in other sequencing methods (e.g., in ion semiconductor DNA sequencing systems). 
       SUMMARY AND DESCRIPTION 
       [0009]    The scope of the present invention is defined solely by the appended claims and is not affected to any degree by the statements within this summary. 
         [0010]    The present embodiments may obviate one or more of the drawbacks or limitations in the related art. For example, the efficiency of sequencings of biopolymers is increased. 
         [0011]    The present embodiments are based on the concept of combining an arrangement for sequencing (e.g., a nanopore arrangement) with principles from image sensor technology. As a result, a number of biopolymers may be simultaneously sequenced in parallel with a low circuit outlay. 
         [0012]    The method according to one or more of the present embodiments correspondingly serves for sequencing at least two biopolymers (e.g., a plurality of identical or different nucleic acids or proteins) including the following acts. For each biopolymer, a sequence signal is picked up by a respective measurement variable pick-up depending on the sequence of the biopolymer. Each sequence signal is transferred in parallel into a shift register, and each sequence signal is buffer-stored by the shift register. The buffer-stored sequence signals are sequentially transferred from the shift register into an evaluation device (e.g., a microprocessor of a computer. The sequence signals are evaluated by the evaluation device. 
         [0013]    In this case, a shift register may be an analog bucket brigade device (e.g., a switching mechanism) in which a plurality of multivibrators connected in series shift along stored content by one multivibrator upon each operating clock cycle according to the bucket brigade principle. One example of a shift register is a CCD image sensor. 
         [0014]    Sequentially transferring each outgoing sequence signal of a measurement variable pick-up includes transferring into a respective multivibrator of a shift register. As a result, the shift register, via multivibrators of the shift register, may receive sequence signals from measurement variable pick-ups connected upstream in parallel. This makes it possible to multiply the number of simultaneously usable arrangements for sequencing (e.g., to multiply the number of simultaneous sequencings). 
         [0015]    In this case, a sequence signal may describe a current intensity or a voltage value. The use of a shift register ensures noise-free and low-loss charge transfer. In addition, a plurality of biopolymers may be sequenced simultaneously, and the sequence signals of the different biopolymers may be “collected” (e.g., detected at the different measurement pick-ups, in parallel or simultaneously). 
         [0016]    In one embodiment of the method, transferring each sequence signal into a shift register, which together with the measurement variable pick-up forms an integrated electrical circuit, is carried out, for example, on a sensor array as microchip. This enables simultaneous sequencing of the biopolymers and simultaneous reading-out of the signals for a number of identical or different biopolymers. In this case, picking up the sequence signal of the first biopolymer may be carried out at the same time as picking up the sequence signal of the further biopolymer. The simultaneous reading-out of the signals is made possible as a result. 
         [0017]    Very fast sequencing is made possible as a result. A sequencing may be carried out by the integrated arrangement in less than one hour, for example. 
         [0018]    Picking up the sequence signal with the aid of a nanopore arrangement including a respective nanopore for sequencing one of the biopolymers constitutes one embodiment of the method. A sequencing method with the aid of a nanopore arrangement enables a better resolution compared with other methods. The respective sequence signal may then describe, for example, a tunneling current that flows in the nanopore when the biopolymer passes through the nanopore. Tunneling current methods have a better resolution in comparison with, for example, measuring a pore resistance on account of high electric field strengths within the nanopore. 
         [0019]    In accordance with a further embodiment, additional amplification of each sequence signal may be achieved by a respective preamplifier before the sequence signal is transferred into the shift register. 
         [0020]    In a further embodiment of the method, sequentially transferring the buffer-stored sequence signals includes singly or multiply amplifying each sequence signal by one or a plurality of output amplifiers. In a further embodiment, such an output amplifier may include one or a plurality of electron multiplying charge-coupled device (EMCCD) stages (e.g., in which an amplifier section is implemented between a shift register and an output amplifier). The EMCCD stages are distinguished by almost lossless signal transfer and multiplication of the signal strength, such that the sequence signals are scarcely attenuated or corrupted. 
         [0021]    Transferring each sequence signal into the shift register may be carried out, for example, at a frequency of between 0.5 kilohertz and 10 kilohertz (e.g., at 1 kilohertz). This transfer frequency range takes account of the characteristic speed of, for example, a base of a nucleic strand when passing through a nanopore, for example, in one millisecond. 
         [0022]    In one embodiment, the outgoing signal of the shift register and an outgoing signal of a further shift register of the same type are transferred in parallel to a collective shift register, which transfers the signals sequentially to the evaluation device. The shift registers are therefore arranged in a comb structure, where the shift registers that receive a respective sequence signal from the measurement pick-ups are connected to the collective shift register like tines. In this case, the collective shift register collects the outgoing signals from the shift registers in multivibrators. 
         [0023]    A sequencing arrangement for detecting and/or sequencing a plurality of biopolymers includes at least one measurement variable pick-up for picking up a sequence signal of one of the biopolymers. The sequencing arrangement also includes a shift register electrically connected to each measurement variable pick-up. The respective advantages of the embodiments are evident in each case from the above description concerning the method. 
         [0024]    In this case, the measurement variable pick-ups and the shift register may be embodied as an integrated electrical circuit (e.g., arranged on a common microchip or silicon wafer). 
         [0025]    The sequencing arrangement according to a further embodiment also includes a plurality of nanopore arrangements having a respective nanopore and a respective measurement variable pick-up for sequencing one of the biopolymers. The respective measurement variable pick-up may be configured to pick up a tunneling current when the biopolymer is present in the nanopore. 
         [0026]    One embodiment of the sequencing arrangement may include a respective preamplifier between each measurement variable pick-up and the shift register. 
         [0027]    In a further embodiment, the sequencing arrangement may include at least one output amplifier for amplifying a sequence signal. The at least one output amplifier is connected downstream of the shift register. This may also include one or a plurality of EMCCD stages, for example. 
         [0028]    In one embodiment, the sequencing arrangement includes at least one further shift register of the stated type. The shift registers are coupled to the evaluation device via a collective shift register. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0029]    Functionally same elements bear the same reference signs in the figures. In the figures: 
           [0030]      FIG. 1  shows a schematic illustration of a sequencing apparatus known from the prior art in longitudinal section; 
           [0031]      FIG. 2  shows a schematic illustration of a sequencing apparatus known from the prior art in cross section; 
           [0032]      FIG. 3  shows a schematic illustration of one embodiment of a device; 
           [0033]      FIG. 4  shows a schematic illustration of a further embodiment of a device; 
           [0034]      FIG. 5  shows a schematic illustration of one embodiment of a device; and 
           [0035]      FIG. 6  shows a circuit diagram concerning a preamplifier in accordance with one exemplary embodiment. 
       
    
    
     DETAILED DESCRIPTION 
       [0036]    A typical construction of a nanopore sequencer  1  from the prior art is shown in longitudinal section in  FIG. 1 . The sequencing device  1  includes a nanopore  2 , which is produced on a silicon carrier wafer  3 , for example. 
         [0037]      FIG. 2  shows a typical nanopore arrangement  10  from above. A nanopore  2  between the electrodes  4  is indicated schematically in cross section in  FIG. 2 . The nanopore  2  is, for example, part of a membrane (not shown in  FIG. 1 ) between the two electrodes  4 . Two tunnel electrodes  4  are fitted directly at the nanopore  2 . The tunnel electrodes are arranged between insulating layers  5  in the example in  FIG. 1 . If a biopolymer  6  (e.g., a DNA molecule), using an applied voltage, is then drawn from the upper reservoir in  FIG. 1  into the lower reservoir through the nanopore  2 , a tunneling current flowing between the two tunnel electrodes  4  is modulated. 
         [0038]      FIG. 3  shows one exemplary embodiment of a sequencing arrangement  11  according to one or more embodiments, on the basis of which one exemplary embodiment of the method will be explained. There are, for example, four nanopore arrangements  10  therein, which may include, for example, a membrane with one nanopore and two electrodes flanking the nanopore, as described in  FIG. 1  and  FIG. 2 . 
         [0039]    A tunnel voltage (“V T ”) is applied to a first tunnel electrode via an electrical connection  12 . When a biopolymer  6  such as, for example, a nucleic acid passes through, a tunneling current flowing between the two tunnel electrodes  4  arises in each nanopore and is modulated as a sequence signal. The tunneling current is picked up by a measurement variable pick-up and transferred into a respective multivibrator  20  of a shift register  16  via an electrical connection  18 . 
         [0040]    The shift register  16  may be embodied as an analog “charge coupled device” (CCD). A shift register  16  has been known to a person skilled in the art previously as an analog shift register or as an image sensor. The shift register  16  is distinguished by almost lossless transfer of charge packets along the shift register  16 . In this case, the charge transfer from individual multivibrators functions according to the bucket brigade principle. In the shift register  16 , an integrated storage device  22  may be connected upstream of each multivibrator  20 . The integrated storage device  22  buffer-stores and optionally amplifies a sequence signal. The respective storage device  22  may include a capacitor. In  FIG. 3 , the arrows within each storage device  22  indicate that the storage device  22  transfers the amplified signal into the multivibrator  20  respectively connected downstream of the storage device  22 . The optionally amplified tunneling current as sequence signal may therefore be integrated in an input stage (e.g., in the storage device  22 ) of the shift register  16  and be accepted at periodic intervals in parallel into the shift register  16  by the activation of a “load” signal (“L”) (method act S 1 ). In this case, transferring a sequence signal into the shift register  16  may be carried out at a frequency of between 0.5 kilohertz and 10 kilohertz (e.g., at 1 kilohertz). 
         [0041]    The content of each storage device  22  may then be shifted serially from the shift register  16  in a known manner with the aid of non-overlapping control signals “φ0”, “φ1” and “φ2” for the shift register  16 . An output amplifier  24  may also amplify the resultant serial AOUT signal (“A”) and make the resultant serial AOUT signal available for further processing (e.g., transfer the resultant serial AOUT signal to an evaluation device  26  such as a microprocessor of a computer). However, the output signal A may also be transferred directly from the shift register  16  to the evaluation device  26 . In other words, the sequence signals are sampled, and the samples, collected by the shift register  16 , are transferred to an individual evaluation device  26 . 
         [0042]    In the example in  FIG. 3 , four nanopore arrangements are shown, but generally an arbitrary plurality of nanopore arrangements  10  connected in parallel are provided. As an alternative to a plurality of nanopore arrangements  10 , a plurality of other arrangements  10  may be provided for generating a sequence signal in the method according to one or more of the present embodiments and in the device according to one or more of the present embodiments (e.g., arrangements  10  for generating a sequence signal including in each case an ion semiconductor DNA sequencing system). The plurality of nanopore arrangements  10  may be arranged, for example, in a sequencing apparatus  1  and includes a measurement variable pick-up for picking up a tunneling current of the nanopore. The sequencing apparatus  1  may likewise include, as shown in  FIG. 3 , the preamplifier or preamplifiers  14  and/or the shift register  16 . In one embodiment, the shift register  16  and the plurality of nanopore arrangements  10  may be integrated within the same circuit (e.g., integrated on one microchip and/or in one sequencing apparatus). The advantage of such an integrated circuit resides in the almost lossless transfer of each sequence signal. Alternatively, however, the stated components may also be arranged in different apparatuses, and these apparatuses may be coupled to one another by electrical connections. 
         [0043]    In one embodiment of the method or of the sequencing arrangement  11 , a sequencing arrangement  11  according to one or more of the present embodiments may also be extended to form, for example, a sensor array or may be integrated within the same apparatus. This is shown in  FIG. 4 . The sequencing arrangement  11  shown in  FIG. 4  is derived from the columnar arrangement from  FIG. 3 . For the sake of clarity, only one of the shift registers  16  connected in parallel and only a few of the nanopore arrangements  10 , preamplifiers  14 , input stages  22 , and multivibrators  20  are identified by reference signs. The construction of a respective shift register  16  and of the respective arrangement connected upstream is evident from  FIG. 3  and the associated description. 
         [0044]    The individual columns of the arrangements (in each case including a plurality of nanopore arrangements  10  and a shift register  16 ) are coupled by a further shift register  16 ′, which accepts the AOUT information of the individual columns once again in parallel into multivibrators  20 ′ of the further shift register  16 ′ and makes the AOUT information available serially at the output  34 . A coupled arrangement, which describes the coupling together of two shift registers, is known to the person skilled in the art from EP 0 303 846 B1. 
         [0045]    A measurement cycle may proceed as follows. First, the sequence signals are accepted into the column shift registers  16  by the activation of a “LoadV” signal (“LV”). The electrical charge of the output cell is then accepted into the row shift register  16 ′ by the activation of the control signal “LoadH” (“LH”). As the next act, the row shift register  16 ′ is shifted out, for example, by the control signals φ0 H, φ1 H and φ2 H for the shift register  16 ′. The data in the column shift registers  16  are then shifted by one position (e.g., by the control signals φ0V, φ1V and φ2V), and the read-out process at the row shift register  16 ′ may be repeated until all the sequence signals are read out. 
         [0046]    In a further exemplary embodiment, which is shown in  FIG. 5 , the sequencing arrangement  11  according to one or more of the present embodiments may be combined with a further output amplifier  24 ′ (e.g., an “electron multiplying CCD stage” (EMCCD)). EMCCD stages are known from camera technology. EMCCD stages are used principally in particularly sensitive camera systems. In each stage  32  of the further output amplifier  24 ′, the number of electrons is increased from stage to stage by impact ionization. Moreover, the sequencing arrangement  11  shown in  FIG. 5  may include a sequencing arrangement  11  in accordance with  FIG. 3  and the associated description. 
         [0047]    By way of example, a transimpedance arrangement, as known from Rosenstein et al., for example, is suitable, in principle, as preamplifier  14 . However, the relatively high stray capacitance of the tunnel contacts may lead to stability problems for the preamplifiers and to an increase in noise. A circuit diagram for a preamplifier  14  in the case of high input capacitances is, for example, a preamplifier  14  depicted schematically in  FIG. 6 . This may involve, for example, a circuit having a “common-base” input stage including a low input resistance and a current mirror  30  with respect to the output. The functioning per se is evident from the circuit diagram. By way of example, the two transistors Q in  and Q er  form the input stage. The signal may be brought to the output via the two current mirror transistors M p1  and M p2 . In the example, I b  is the bias current, R f  is a feedback resistance, and R o  is an output resistance. I in  represents an input current, and Vow represents the output voltage. 
         [0048]    The exemplary embodiments presented above illustrate a principle of the present embodiments (e.g., combining an arrangement for sequencing, such as a nanopore arrangement, with principles from image sensor technology). 
         [0049]    As a result, an integrated arrangement in which, for example, an array of nanopores  4  is combined with, for example, amplifiers  14 ,  24  and a read-out unit  26 , such that the possible throughput of one or a plurality of arrangements for sequencing is significantly increased. 
         [0050]    The elements and features recited in the appended claims may be combined in different ways to produce new claims that likewise fall within the scope of the present invention. Thus, whereas the dependent claims appended below depend from only a single independent or dependent claim, it is to be understood that these dependent claims may, alternatively, be made to depend in the alternative from any preceding or following claim, whether independent or dependent. Such new combinations are to be understood as forming a part of the present specification. 
         [0051]    While the present invention has been described above by reference to various embodiments, it should be understood that many changes and modifications can be made to the described embodiments. It is therefore intended that the foregoing description be regarded as illustrative rather than limiting, and that it be understood that all equivalents and/or combinations of embodiments are intended to be included in this description.