OPTICAL CONTROLLING OF A CHEMICAL REACTION

Abstract

The present invention relates to a device (100) and a method for optically controlling a chemical reaction in a reaction chamber (149) comprising a reagent fluid (114). In a preferred embodiment, the chemical reaction comprises a nucleic acid sequencing on a wiregrid. Based on strong optical confinement of excitation light (110) and of cleavage light (112), the sequencing reaction can be read-out. Stepwise sequencing is achieved by using nucleotides with optically cleavable blocking moieties. After read-out the built in nucleotide is deblocked by cleavage light through the same substrate. This ensures that only bound nucleotides will be unblocked. In order to avoid overheating by cleavage light, the reagent fluid is circulated along the surface of the substrate (101).

Claims

1. A device for optically controlling a chemical reaction in a reaction chamber comprising a reagent fluid, said device comprising: a substrate for binding at least one molecule on a first surface of the substrate, wherein said first surface is a wall of the reaction chamber; an optical arrangement configured to direct cleavage light to the substrate to optically induce a photochemical cleavage reaction; a circulation arrangement for circulating the reagent fluid in the reaction chamber, wherein the substrate is configured as a wiregrid.

2. The device according to claim 1, wherein the reagent fluid is circulated along the first surface of the substrate.

3. The device according to claim 1, wherein the circulation arrangement comprises a pumping element for actively pumping reagent fluid and/or a cooling element for cooling reagent fluid.

4. The device according to claim 1, wherein the circulation arrangement comprises at least one pneumatically driven actuator.

5. The device according to claim 1, wherein the circulation arrangement is adapted to synchronize the circulation of the reagent fluid with the irradiation of cleavage light.

6. The device according to claim 1, wherein the chemical reaction comprises a nucleic acid sequencing, in particular an iterative stepwise reaction to determine a sequence of a nucleic acid by synthesis.

7. The device according to claim 1, wherein the optical arrangement is configured to direct excitation light of at least a first excitation wavelength .sub.Ex1 to the substrate to excite a fluorescent label of a first nucleotide incorporated into the molecule bound on the first surface of the substrate, wherein the optical arrangement is further configured to receive and detect fluorescent light emitted by the fluorescent label of the first nucleotide incorporated into the bound molecule.

8. The device according to claim 7, wherein the cleavage light, preferably UV light, has a cleavage wavelength .sub.CL to optically induce a photochemical cleavage reaction at the first incorporated nucleotide to cleave a blocking moiety and the fluorescent label away from the first incorporated nucleotide.

9. The device according to claim 1, wherein the substrate is configured to confine the excitation light and is configured to provide for an evanescent wave of the excitation light at the first surface of the substrate, and/or wherein the substrate is configured to confine the cleavage light and is configured to provide for an evanescent wave of cleavage light at the first surface of the substrate.

10. The device according to claim 8, the device further comprising: the molecule which is bound to the first surface of the substrate, a solution with a plurality of nucleotides and an enzyme, wherein the nucleotides respectively comprise the blocking moiety, wherein the blocking moiety is configured to block a synthesizing activity of the enzyme when the respective nucleotide is incorporated into the molecule bound to the first surface.

11. A method for optically controlling a chemical reaction in a reaction chamber comprising a reagent fluid, said method comprising the following steps: providing a substrate with a molecule bound on a first surface of the substrate, wherein said first surface is a wall of the reaction chamber, irradiating the substrate with cleavage light of a cleavage wavelength .sub.CL, preferably UV light, by an optical arrangement and thereby optically inducing a photochemical cleaving reaction, circulating the reagent fluid in the reaction chamber.

12. The method according to claim 11, further comprising circulating the reagent fluid in the reaction chamber along the first surface of the substrate and/or the active pumping and/or the cooling of the reagent fluid.

13. The method according to claim 11, wherein the circulation of the reagent fluid is synchronized with the irradiation of cleavage light.

14. The method according to claim 11, wherein the intensity of the cleavage light is larger than about 0.1 mW/cm.sup.2.

15. The method according to claim 11, further comprising the steps: irradiating the substrate with excitation light of at least a first excitation wavelength .sub.Ex1 by an optical arrangement and thereby optically exciting a fluorescent label of a first nucleotide which is incorporated in the bound molecule on the substrate, confining the excitation light by the substrate thereby providing for an evanescent wave of the excitation light by the substrate at the first surface of the substrate, receiving and detecting fluorescence of the excited fluorescent label of the first incorporated nucleotide by the optical arrangement, irradiating the substrate with the cleavage light and thereby optically inducing a photochemical cleaving reaction at the first incorporated nucleotide, and confining the cleavage light of the cleavage wavelength .sub.CL by the substrate thereby providing for an evanescent wave of the cleavage light by the substrate at the first surface of the substrate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Exemplary embodiments of the invention will be described in the following drawings.

[0052] FIG. 1 schematically shows a first device according to an exemplary embodiment of the invention.

[0053] FIG. 2 schematically shows a second device according to an exemplary embodiment of the invention.

DETAILED DESCRIPTION OF EMBODIMENTS

[0054] FIG. 1 depicts a device 100 for optically controlling a chemical reaction, in this case particularly an iterative stepwise reaction to determine a sequence of a nucleic acid by synthesis. The device comprises a substrate 101 for binding at least one molecule 102 on the first surface 103 of the substrate. The molecule 102 which is bound on the first or front surface 103 of the substrate 101 can for example be a fragment of a DNA. The first surface 103 constitutes a wall or border of a reaction chamber 149 in which a fluid to be processed (here a solution 114 that is described in more detail below) can be accommodated. The reaction chamber is typically a part of a larger (micro-) fluidic device or cartridge that is not shown in more detail.

[0055] Furthermore, the optical arrangement 104 is shown in FIG. 1. FIG. 1 schematically shows that the optical arrangement is configured to direct excitation light 110 of for example the first excitation wavelength .sub.Ex1 to the substrate. Furthermore, four different nucleotides are schematically shown and are depicted with reference signs 109, 116, 117 and 118. For example, a first nucleotide 109 is shown as Thymine, T. The nucleotide 109 comprises a blocking moiety 119. Furthermore, the blocking moiety 119 comprises the first fluorescent label 105. In an analog way, second nucleotide 116 is schematically depicted in FIG. 1, from which can be gathered that also a blocking moiety 119 and the second fluorescent label 106 are comprised. The third nucleotide 117 comprises also a blocking moiety and a third fluorescent label 107. Additionally, the fourth nucleotide 118 is schematically depicted which comprises also a blocking moiety and a fourth fluorescent label 108. However, sample 114 may comprise a much larger plurality of such nucleotides, and nucleotides 109, 116, 117 and 118 are shown here merely as a symbolic depiction.

[0056] Furthermore, FIG. 1 shows a solution 114 which fills the reaction chamber 149 and in which the nucleotides and the enzyme 115 are comprised. In case one of the shown four nucleotides is incorporated in the bound molecule 102, the presented device 100 provides for the following advantages. The optical arrangement is configured to receive and detect fluorescence light emitted by the fluorescent label of the first nucleotide incorporated into the bound molecule 102.

[0057] As can further be gathered from FIG. 1, the optical arrangement is configured to direct cleavage light 112 of cleavage wavelength .sub.CL to the substrate. This allows for optically inducing a photochemical cleavage reaction at the first incorporated nucleotide to cleave the respective fluorescence label from the first incorporated nucleotide. Furthermore, the substrate 101 is configured to confine excitation light such that an evanescent wave of the excitation light at the first surface of the substrate is created. Moreover, the substrate is configured to confine also the cleavage light such that an evanescent wave of the cleavage light at the first surface of the substrate is created.

[0058] In the embodiment of FIG. 1, the substrate 101 is configured as a wiregrid 113 for the excitation light 110 and for the cleavage light 112. Therefore, the wiregrid 113 comprises a regular pattern, like for example a regular metal wire structure. As can be gathered from FIG. 1, slit-like openings are provided between the regular patterns, in which openings the bound molecules 102 are immobilized at the first surface 103 of the substrate 101.

[0059] Furthermore, FIG. 1 depicts a processing unit 120 which comprises a computer-readable medium 121 on which a computer program element 122 is stored. Said program element 122 is adapted to instruct the processing unit 120 to further instruct the device 100 to perform the above and below described method for optically controlling an iterative stepwise reaction to determine a sequence of a nucleic acid by synthesis. The device 100 of FIG. 1 is configured to stepwise and optically induce the incorporation of nucleotides 109, 116, 117, 119 with a sequence, which corresponds to the sequence of nucleotides of the bound molecule 102. In case the molecule 102 is a DNA fragment, the nucleotides comprised by the sample 114 are incorporated into molecule 102 in a sequence that corresponds to the nucleotide sequence of molecule 102.

[0060] The device is further configured to base the determination of the sequence of the incorporated nucleotides on the received and detected response fluorescence light emitted by the fluorescent label of the respective incorporated nucleotide. Therefore, the presented device 100 of FIG. 1 firstly ensures that only nucleotides are read-out by the excitation light 110, which nucleotides are incorporated into a bound molecule 102 by the use of an evanescent wave of the excitation light. Secondly, the device 100 of FIG. 1 ensures that only bound nucleotides will be unblocked by the cleavage light which avoids a bleaching and loss of function of nucleotides that are not yet contained i.e. incorporated by the molecule 102. Consequently, the detected fluorescence signal 100 may be seen as the light 111, is highly reliable for the determination of the sequence of the nucleic acids.

[0061] Consequently, the cost and speed of the nucleic acid sequencing, like for example the DNA sequencing performed with the device 100 of FIG. 1 are both improved. Less reagents and enzymes are necessary as no washing step is needed. The device of FIG. 1 shows a simplification and cost reduction of sequencing. The presented device 100 of FIG. 1 allows for a new process combination by allowing an assemble-based easy read-out without any washing step, meaning a single reagent filling for all reads. The blocking moieties used within the exemplary nucleotides 109, 116, 117, 118 may for example be a photo-cleavable 3-unblocked reversible terminator. However, also other blocking moieties, using for example steric hindering, may be used to reach the desired and above described effects.

[0062] Furthermore, the optical arrangement 104 as shown in FIG. 1 may be configured to provide the irradiated cleave light with an intensity such that the cleaving reaction time t.sub.cleavage is smaller than the time it takes to incorporate the second nucleotide into the molecule 102. As the cleaving reaction time t.sub.cleavage depends on the intensity of the irradiated cleavage light, FIG. 1 may provide for a selected combination of nucleotides with a specific blocking moiety and a configuration of the optical arrangement regarding the intensity of the cleavage light. In other words, the intensity of the cleavage light of the device of FIG. 1 is adapted such that for the used combination of nucleotides and blocking moieties the cleaving reaction time t.sub.cleavage is smaller than t.sub.incorporation.

[0063] If desired, additionally or alternatively, the following set-up of device 100 may be provided to the user. If the reagent fluid is stationary and movement of molecules driven by diffusion, then the residence may be seen as an average residence time in the spot of cleavage light of a non-incorporated nucleotide. An optical arrangement may further be configured to provide the irradiated cleavage light with an intensity such that t.sub.cleavage is smaller than t.sub.residence. Consequently, no degradation of free and unbound nucleotides due to an undesired cleavage reaction happens. Thus, by configuring the device such that t.sub.cleavage is smaller than t.sub.residence the probability that a non-incorporated nucleotide is affected by cleaving is reduced or eliminated. In other words, to avoid cleavage reactions in the bulk the average residence time of the molecules in the evanescent field of the wiregrid should be smaller or much smaller than the reaction time required for cleavage at the pertinent intensity. With a depth of the evanescent field of the order of 25 nm or less and a diffusion coefficient of the nucleotide of the order of 1e10 m.sup.2/s the time it takes for the molecule to diffuse in and out the evanescent field can be estimated as: (5e8 m)2/1e10=25 s. Depending on the illumination time required for unblocking the bound molecules the probability of damage can be derived. Assume an illumination time of 0.1 s this would be 1:4000, with an illumination time of 10 ms it would be 1:400, etc.

[0064] Likewise the total damage is proportional to the volume fraction in the evanescent field over the total volume of reagent solution. With a chamber height of 100 m the ratio is 1:4000. This means that in the worst case of damaging all molecules in the evanescent field only 0.025% of the molecules will be damaged. With a read length of 100 finally 2.5% of the molecules in solution would be damaged (worst case) which is still acceptable from a sequencing point of view.

[0065] The above considerations are valid for a stationary fluid in the reaction chamber 149. If the reagent fluid is however circulated in the reaction chamber (as explained in more detail below), movement of the molecules is dominated by active pumping rather than by diffusion. To effect cooling by fluid circulation, it is desirable to change the fluid in the excitation volume repeatedly, for example between 10 and 100 times per interval between the start of two pulses of cleavage light. Under these circumstances the balance between the cooling effect one would like to achieve and not cleaving too many unbound nucleotides has to be considered (as cleaved unbound nucleotides incorporated into the DNA cannot be detected because they no longer have a fluorophore identifying the base). In particular, the residence time of the liquid in the excitation volume should be shorter than the UV cleavage time as otherwise no additional cooling is achieved (during UV cleavage).

[0066] If the volume which is excited by the cleavage light is assumed to be a cylinder with a diameter of about 100 nm and a height of about 25 nm, a very small volume of about 210(8) l or 0.02 pl results (in comparison to a total volume of the solution of typically about 1-5 ml). Hence one has to consider the concentration of the labeled nucleotides and the refresh rate, which could become between about 2 to 10 times replacing the volume, possibly between about 2 to 5 times. So in practice for a 5 replacement on a total of 10 ml solution, there is a factor of 10.sup.+11 between the volume irradiated with UV blocking light and the total volume per spot.

[0067] In the following, information for using the device of FIG. 1 (and FIG. 2) is provided. For an improved synchronization the unblocking step should be carried out as fast as possible, i.e. with the highest intensity possible. This can be achieved by focusing the UV-light with a lens and scanning the surface by moving the lens or the substrate. The unblocking step is carried out after reading the sequencing step. This reading can be carried out by scanning a focused beam or step-and-scan with field illumination. In a preferred embodiment the read scanning can be coupled to the unblocking scanning by integrating both light beams in a single actuator, possibly even in a single lens by aligning the light beams. Alternatively, two lenses can be integrated in a single stage or two separate stages can operate synchronously. This can also be implemented in the step and scan read approach, in which the UV-step is also carried out in a step and scan mode by illuminating the same field as the reader. The preferred embodiment will depend on the available UV light source and its power. One can also envision a single flash of UV for the total surface if enough power is available and/or the area of the sequencing surface is limited. In view of the reaction rate for the base incorporation for the sequencing reaction the local UV illumination time should be well below 1 minute.

[0068] A single fluid sequencing using a wiregrid as well as single molecule sequencing has been described above. The approaches may use so called 3-unblocked reversible terminators in which a flash of UV light is needed to de-block the nucleotide so that the next labeled nucleotide with a fluorophore attached can be incorporated by the polymerase. Reading out the color of the incorporated nucleotide allows the base incorporated to be determined and hence sequencing to be done.

[0069] In the described procedures high intensity UV light is needed. Typical intensity values range from about 4 mW/cm.sup.2 to about 1 W/cm.sup.2. This corresponds to a considerable amount of energy that may cause heating in the wiregrid and the buffers containing the reagents.

[0070] In order to improve the system performance and avoid overheating of the wiregrid/local liquids and even of the cartridge, it is proposed to circulate the reagent liquid (here the buffer and the required enzymes/nucleotides) by pumping them, for instance by using a pneumatically driven pumping of the liquid of a pneumatically operated cartridge design. This will produce a cooling effect and help to avoid local overheating.

[0071] The aforementioned proposal is realized in the device 100 of FIG. 1 by a circulation arrangement 150 which is schematically indicated as a channel 151 connecting opposite ends of the reaction chamber 149. In the illustrated embodiment, the channel 151 comprises a pumping element 152 by which the fluid in the channel 151 can actively and controllably be pumped (in the direction shown be the arrow). This induces a forced circulation in the reaction chamber 149 with a flow of the reagent fluid along the surface 103. Thus the molecules 102 are always surrounded by the chemicals they need while excess heat, particularly heat generated by cleavage light, is carried away from the surface to avoid overheating.

[0072] The aforementioned excess heat will typically be released to the environment by the reagent fluid during its circulation through other components of the cartridge. In order to assist this process, a cooling element 153 acting as a heat sink may be provided. This may for example be an area or region with close thermal contact to the environment to allow for a cooling effect by the ambient atmosphere. Additionally or alternatively, the cooling element 153 may comprise some active cooling unit such as a Peltier element.

[0073] In a preferred embodiment, the (active, controlled) circulation of reagent fluid by the circulation arrangement 150 may be synchronized with the generation of heat at the surface 103, particularly with the irradiation of cleavage light 112. The active circulation may for example be limited to the intervals of UV de-blocking pulses.

[0074] Accordingly a sequencing system is provided in which sequencing is done using a wiregrid in combination with circulating the buffers containing the reagents to avoid overheating the system while de-blocking using UV light.

[0075] FIG. 2 shows a device 100 which is configured to optically control an iterative stepwise reaction to determine a sequence of a nucleic acid by synthesis. Similar to FIG. 1, a wiregrid substrate 113 is shown on which a plurality of molecules 102 are immobilized, i.e. are bound. As can be seen from FIG. 2, a regular pattern 214 provides for slit-like openings 215 in which the molecules 202 are bound on the first surface 103. The substrate comprises several adjacent binding positions 209, 210, 211 and 212 for binding molecules to the first surface along a first direction 213. Said binding positions may be seen as spots which can be covered with clones of identical molecules, such that the optical signal, which is generated, can be increased. The substrate 101 then provides for an array of such spots, i.e. of such binding positions, with respectively different clones. This may enhance the throughput. Both devices 100 of FIGS. 1 and 2 allow a nucleic acid sequencing with only one liquid, thereby avoiding the need to provide for washing steps in which the solution liquid is changed.

[0076] Furthermore, the optical arrangement 104 comprises five different light sources 201 to 205. The light sources 201 to 204 may be seen as excitation light sources in order to provide for four different excitation wavelength .sub.Ex1 to .sub.Ex4 as described previously. The light source 205 provides for cleavage light with a wavelength .sub.CL. For example, the light source 205 may emit UV light. Reference numeral 206 symbolically depicts a switching device which allows the optical arrangement 104 to switch between the five wavelengths .sub.Ex1 to .sub.Ex4 and .sub.CL. Furthermore, the light emitted by at least one of said light sources 201 to 205 is directed towards the polarization filter 200. Furthermore, a dichroic mirror 207 is shown which transmits the emitted light of the light sources 201 to 205 towards the substrate 101. After a fluorescent label has been excited by an evanescent wave of excitation light (at least one of the wavelengths .sub.Ex1 to .sub.Ex4), the fluorescence photons emitted by the fluorescent label or labels are directed towards the dichroic mirror 207 and are directed towards fluorescence detector 208. As can be seen from FIG. 2, the optical arrangement 104 may be scanned along the direction 213. Consequently, the device 100 of FIG. 2 is configured to perform an optical scan by moving the substrate 101 and the optical arrangement 104 relative to each other along the first direction 213. Consequently, the device allows to perform the optical scan such that each binding position is firstly irradiated with the excitation light and subsequently and secondly is irradiated the cleavage light of the cleavage wavelength in a movement along the first direction 213. The unblocking step, using the cleavage light, can thus be carried out after reading the fluorescence of the excited incorporated nucleotides.

[0077] FIG. 2 further indicates a circulation arrangement 150 as described above with respect to FIG. 1 which allows for a controlled circulation of reagent fluid through the reaction chamber 149 and along the reaction surface with the molecules 102, 209, 210, 211, 212. It should be noted that the flow of reagent fluid may in general have any orientation with respect to the wiregrid 113. It may for example be parallel to the direction 213, as shown, or perpendicular or have any other orientation that is convenient in the case at hand.

[0078] Further details and other exemplary devices and methods may be found in the WO 2013/105025 A1 which is entirely incorporated into the present text by reference.

[0079] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word comprising does not exclude other elements or steps, and the indefinite article a or an does not exclude a plurality. A single processor or other unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. A computer program may be stored/distributed on a suitable medium, such as an optical storage medium or a solid-state medium supplied together with or as part of other hardware, but may also be distributed in other forms, such as via the Internet or other wired or wireless telecommunication systems. Any reference signs in the claims should not be construed as limiting the scope.