Patent Application
20250002975
The technical field of the invention is detection of biomolecules via optical transduction performed with nanowires.
Devices allowing biomolecules to be detected may be based on optical detection. The biomolecules to be detected are labelled beforehand using a luminescent label, such as a fluorophore or quantum-dot label. Detection is achieved via a photodetector array such as a CCD or CMOS imager or an array of avalanche photodetectors (APDs).
A widespread application of fluorescent labelling is sequencing, in particular so-called SBS sequencing (SBS standing for Sequencing By Synthesis). According to this method, a sequence of single-stranded nucleotides, placed downstream of a primer, is amplified to form a cluster. During sequencing, a complementary sequence is gradually synthesized, by successively adding nucleic bases complementary to those forming the studied sequence. The complementary bases are added one by one, in respective cycles. After each addition of a complementary base, an image of the cluster is taken. All or some of the complementary bases are labelled with a fluorescent label. Observing the fluorescence in each cycle allows the complementary base added in said cycle to be identified. Thus, by taking a succession of fluorescence images, it is possible to decode the examined nucleotide sequence.
The fluorescence is detected by a photodetector, usually a photodetector array. However, the detector arrays of such photodetectors generally have a spatial pitch greater than 1 μm. By spatial pitch, what is meant is the distance between two adjacent pixels. Such a spatial pitch is considered to be too large to obtain a device allowing a large number of detections in parallel. These devices also have a limited sensitivity: it is necessary to provide sufficient amplification to obtain a sufficient number of identical sequences at the studied cluster, to obtain an exploitable signal.
Certain biosensors are based on detection of charges carried by the target biomolecules to be detected. It is a question for example of field-effect transistors. This type of device allows rapid detection, with a good sensitivity. In addition, CMOS technology (CMOS standing for Complementary Metal-Oxide-Semiconductor) allows devices with a large number of sensors to be manufactured. It is thus possible to carry out a large number of analyzes in parallel. However, this type of sensor may be sensitive to environmental parameters affecting the sample, for example pH or temperature or interactions with non-targeted biomolecules or ions inducing detection errors. Use of this type of biosensor in DNA sequencing applications (DNA standing for DeoxyriboNucleic Acid) requires a certain redundancy in the measurements, so as to increase their robustness. DNA sequencers based on nanopores have the same drawbacks.
Nanowire-based photonic sensors have been developed for the purpose of detecting DNA fragments. This is for example the case in the publication Sing, et al., “Silicon nanowire optical rectangular waveguide biosensor for DNA hybridization”, IEEE Photonics Technology Letters, 2018, 30 (12) 1123-1126. In this publication, DNA hybridization is detected by detecting a variation in refractive index during hybridization.
The publication Irrera, et al., “New generation of ultrasensitive label-free optical Si nanowire-based biosensors”, ACS Photonics 2018, 5 (2) 471-479 describes a nanowire-based biosensor used to detect CRP (C-reactive protein) in human serum.
Applications related to DNA sequencing require fluorescence coding, to differentiate the added bases. In applications employing a sensor, coding is achieved either through the emission wavelength, or through a light intensity in the image acquired by the photodetector.
The inventors have designed a device for analyzing a sample that is compact and sensitive, allowing advantage to be taken of the fluorescence-detecting capabilities of nanowires. The device may allow simultaneous or sequential analysis of various biomolecules. The device makes it possible to benefit from the detection sensitivity and compactness of nanowires, while allowing differentiated detection of nucleic bases labelled with fluorescent labels.
A first subject of the invention is a device for identifying a fluorescent label, the fluorescent label being configured to emit fluorescence light in a fluorescence spectral band, the device comprising:
According to one possibility, the device comprises a light source configured to emit excitation light in the excitation spectral band of the fluorescent label.
According to one possibility:
According to one possibility, each nanowire comprises:
According to one possibility, at least one nanowire comprises a first portion and a second portion, which are separated by the junction, the first portion being formed from an n-doped semiconductor, the second portion being formed from a p-doped semiconductor, the junction forming a homojunction or a heterojunction.
According to one possibility, at least one nanowire comprises a first portion and a second portion, which are separated by the junction, the first portion being formed from a semiconductor, the second portion, adjacent to an electrode, being formed from a metal, the junction forming a Schottky junction.
According to one possibility, the first portion and the second portion extend, along the transverse axis, on either side of the junction.
According to one possibility, the second portion encircles the first portion, around the transverse axis.
According to one possibility, the functionalization surface is segmented into various capture sites, each capture site being configured to capture one strand forming a chain of oligonucleotides.
According to one possibility:
According to one possibility, the device comprises a plurality of nanowires, extending between the same first electrode and the same second electrode, the nanowires forming a nanowire cluster.
According to one possibility, the device comprises a plurality of nanowire clusters spaced apart from one another, such that one nanowire of a cluster is closer to another nanowire of said cluster than to another nanowire of another cluster, the nanowires of a given cluster extending between the same first electrode and the same second electrode.
According to one possibility, the device comprises a plurality of nanowires, the device being such that:
A second subject of the invention is a method for identifying a fluorescent label using a device according to the first subject of the invention, the fluorescent label being capable of emitting fluorescence light, in the detection spectral band, when it is illuminated by excitation light, the functionalization surface being configured to capture a strand of nucleic acid, the method comprising:
Step b) may comprise amplification of each captured strand of nucleic acid.
According to one possibility, steps b) to g) are carried out at various capture sites distributed over the functionalization surface.
According to one possibility, step f) comprises:
According to one possibility:
According to one possibility, step f) comprises estimating a time derivative of the detection signal.
According to one possibility, step f) comprises detecting an intensity level or integrating the detection signal during at least one predetermined time period. According to one possibility:
According to one possibility:
According to one possibility, following step g), the method comprises a step h) of identifying the hybridized nucleic acid base.
According to one possibility:
The invention will be better understood on reading the description of the examples of embodiment presented, in the remainder of the description, with reference to the figures listed below.
The device comprises a substrate 10, forming or comprising at least one first electrode 11c. In the example shown in
Nanowires 30 are formed on the substrate 10, and more precisely from the first surface 11. The first surface 11 lies in a plane PXY. The plane PXY is defined by a longitudinal axis X and a lateral axis Y. The axes X and Y are secant, and preferably perpendicular to each other. The nanowires extend parallel to a transverse axis Z secant to the plane PXY. In the embodiments described below, the transverse axis Z is perpendicular to the plane PXY. The first surface 11 is conductive at least at the intersection with each nanowire 30. The entirety of the first surface 11 may be conductive.
According to other configurations, the nanowires may be inclined and not perpendicular to the plane PXY. For example, if the crystal orientation of the material forming the substrate 10 is (001), the nanowires may grow in a (111) direction, and therefore slanted with respect to the plane PXY.
The nanowires 30 preferably have a diameter between 1 nm and 500 nm and a height, along the transverse axis Z, between 300 and 1000 nm, or even 10000 nm. The nanowires 30 may be synthesized directly on the substrate 10, as described with reference to
The nanowires 30 extend, from the first surface 11, to a second surface 21 bounding a multilayer structure 20. Just like the first surface 11, the second surface 21 is conductive at least at the intersection with each nanowire 30. In the example shown in
Each nanowire is formed from one or more semiconductors, and optionally from a metal. Each nanowire comprises a junction 33. In the example shown, the junction 33 is a homojunction: each nanowire comprises a first portion 31, adjacent to the first surface 11, and a second portion 32, adjacent to the second surface 21. The first and second portions are formed from the same semiconductor, but have different respective dopings: thus, the first portion 31 and the second portion 32 are formed from the same semiconductor doped n and p or p and n, respectively. In the example shown, the first portion 31 is formed from p-doped gallium arsenide (GaAs) and the second portion is formed from n-doped GaAs. The interface between the two portions forms the p-n junction 33.
Alternatively, the junction 33 may be formed at a metal placed on or in contact with the surface 21, so as to establish a semiconductor-metal Schottky junction.
Each nanowire comprises at least one semiconductor chosen from the materials of columns Ill and V, usually designated by the term III-V materials, GaAs for example. It may preferably be a question of a material from column III and of arsenic, indium arsenide (InAs) for example. In the example shown, the nanowires 30 are formed from GaAs. Other semiconductors may be envisioned, for example, and non-limitingly, Si, InGaAs, AlGaAs, InGaP, InGaN, GaN, ZnSe, ZnS, ZnO, ZnCdO, ZnTe, CdSe, Ge, GeSn.
Between the first surface 11 and the second surface 21, the nanowires 30 are embedded in an encapsulation layer 15 formed from an insulating material. The encapsulation layer 15 may be formed from a material such as polymethyl methacrylate (PMMA) or benzocyclobutene (BCB), or such as a spin-on glass (SoG), spin-on glasses mainly consisting of silicon oxides and other chemical additives endowing them with specific properties such as adhesion and thermal stability. The encapsulation layer 15 may be deposited by spin coating.
The encapsulation layer 15 is preferably formed following growth of the nanowires, prior to deposition of a second conductive layer 22, bounded by the second surface 21, and intended to form second electrodes 21c. The second electrodes 21c are formed, on the second surface 21, from the conductive layer 22. The conductive layer 22 may be structured so that, on the second surface 21, a plurality of second electrodes 21c are electrically insulated from one another. Thus, the conductive layer may comprise apertures or insulating materials delineating the electrodes 21c. This allows differentiated detection at each nanowire.
Apart from the conductive layer 22, the multilayer structure 20 comprises an interface layer 23, adjacent to the conductive layer 22. The interface layer 23 is transparent in the detection spectral band. The interface layer 23 may for example be formed from a layer of a polymer, polymethyl methacrylate (PMMA) for example. The interface layer 23 is electrically insulating, in particular at the interface with the conductive layer 22. The interface layer 23 is preferably planar.
The interface layer 23 is intended to form an interface between the conductive layer 22, forming the electrodes 21c, and the sample 2. Thus, the interface layer 23 lies between the conductive layer 22 and the sample 2. An important aspect of the device is that the sample does not make direct contact with the nanowires. It is isolated from the latter by the interface layer 23.
The interface layer 23 is for example a thin layer formed from SiO2 or PMMA. The surface of the interface layer 23 intended to make contact with the sample is a surface, called the functionalization surface 25, that is functionalized by biological capture species 26. It is important for the interface layer 23 to be formed from a material having as low an autofluorescence as possible in the detection spectral band.
The functionalization surface 25, which forms an interface between the multilayer structure 20 and the sample 2 to be analyzed, is intended to be functionalized by capture species 26. By capture species, what is meant is a species configured to capture a biomolecule of interest, and more precisely a nucleotide sequence to be analyzed.
According to one possibility, the capture species is formed from oligonucleotides configured to graft to the nucleic acid to be analyzed. It may in particular comprise a chain of nucleotide bases complementary to all or some of the adapters bound to the strands to be characterized. The capture species 26 and each strand of oligonucleotides to be analyzed, or more precisely an adapter of each strand of oligonucleotides to be analyzed, then bind through hybridization. The interface layer 23 may be nanostructured, to form nanowells 27 at the interface between the multilayer structure 20 and the sample to be analyzed. The diameter or largest diagonal of the nanowells may be between 70 nm and 700 nm. The nanowells 27 may for example be arranged in a matrix array, or, more generally, in a predetermined pattern. The functionalization surface is then functionalized in each nanowell 27, the spaces between each well not being functionalized. The nanowells may be formed by thinning the interface layer 23 locally.
According to one possibility, shown in
More generally, the functionalization surface 25 may be functionalized in a predetermined functionalization pattern. Outside of the functionalization pattern, the functionalization surface is not functionalized
Functionalization may be achieved via a treatment of the functionalization surface 25, for example a plasma/oxygen surface treatment. When the interface layer 23 is formed from PMMA, a plasma/oxygen treatment makes it possible to form carboxyl functions. The capture species 26 may be grafted onto the functionalization surface by covalent bonding. To this end, the capture species comprise a function, for example an amine function, so as to form a covalent bond with the functions of the functionalization surface 25. It may for example be a question of a bond obtained by grafting a thiol function, of the capture species, to an amine function present on the functionalization surface.
According to one possibility, each sequence to be analyzed comprises a thiol function, for example at 3′ end, so as to form a covalent bond with the functionalization surface. In this case, the functionalization surface acts as capture species.
As shown in
As described with reference to
The first and second surfaces may be structured, and comprise various electrodes 11c, 21c that are insulated from one another. In
A plurality of rows and/or a plurality of columns may be biased simultaneously or successively. The detection circuit 40 comprises:
The functionalized portions of the functionalization surface form capture sites for the nucleotide sequences to be characterized.
As indicated above, each biological capture species 26 is configured to capture a biological species of interest, in particular a nucleotide sequence to be analyzed.
Preferably, after a nucleotide sequence has been captured at a capture site, said sequence is amplified, so as to obtain, at the same capture site, a plurality of replicas that are identical, excluding amplification errors, to the captured nucleotide sequence.
The amplification of each captured sequence may be bridge amplification, usually implemented in devices sold by the company Illumina. When bridge amplification is used, the functionalization surface bears capture species configured to graft to the adapters of the captured sequences.
Generally, sequencing comprises a plurality of cycles, in which complementary bases are gradually hybridized along each sequence to be characterized. In a known manner, in each cycle, the sequences to be characterized are immersed in a reaction medium containing bases labelled with a fluorescent label and a hybridase. Hybridization takes place gradually along each strand, base by base, in a predetermined direction, for example from the free end to the end bound to the capture species.
According to one possibility, the fluorescent labels are coupled to a terminator, this preventing hybridization of two consecutive bases in the same cycle. The reaction medium then contains a mixture of bases, each base of a given type being labelled with the same fluorescent label. Two bases of different types are labelled with different fluorescent labels, respectively. Following each hybridization, the fluorescent label is detected after rinsing, this allowing the base hybridized in the cycle to be identified. Following detection, at least the terminator undergoes cleavage. The fluorescent label may also undergo cleavage. The medium is then rinsed and another cycle is initiated. The cycles are repeated until the sequences grafted at the various capture sites have been completely sequenced. Among the four different bases employed, one base may not be labelled.
According to another possibility, in each cycle, the sequences are successively immersed in baths, each bath containing a single type of base. Each base of a given type is labelled with the same fluorescent label. According to this possibility, it is possible, but not essential, for two bases of different types to be labelled with different labels, respectively. Specifically, each type of base is identified by each bath. Between each bath, a rinse is carried out, and fluorescence is detected at each capture site. According to this possibility, the fluorescent labels do not necessarily comprise terminators. Thus, a plurality of identical successive bases may be hybridized with complementary bases of the reaction medium. This results in a fluorescence signal the intensity of which increases as a function of the number of successive bases hybridized.
Following detection of a fluorescence signal, the fluorescent labels labelling each hybridized base in the cycle may be cleaved. As described below, cleavage is not essential.
Thus, at each capture site, each cycle comprises:
Between each cycle, the reaction medium is washed and renewed. Each cluster is excited by a light source 5, in an excitation band of the fluorescent label or of each fluorescent label. The light source is configured to emit excitation light for a short time, of the order of one ns. It may for example be a question of a laser light source. This makes it possible to generate, at the level of each cluster, a fluorescence signal, which depends on the added base (A, C, T or G in the case of a DNA strand).
An important aspect of the invention, described below, consists in translating the fluorescence signal into an electrical signal dependent on the fluorescent label, this making it possible to obtain, in a cycle, an electrical signal dependent on the base added in the cycle.
This approach is substantially different from the sequencing approach based on formation of an image of the sample, each base being identified by a colour code and/or by an intensity level.
The invention takes advantage of the ability of a nanowire to detect an optical signal, in a predefined spectral band, and to convert the optical signal into an electrical detection signal. Thus, the device 1 is based on optical detection of hybridization of a base to a sequence to be analyzed, inducing an electrical response from the device.
The reaction medium 2 is placed in a fluidic chamber 4, in contact with the interface layer 23. The sequences to be characterized and the reaction medium are isolated from the nanowires 30 by the interface layer 23. In this example, the configuration is that in which the reaction medium contains various types of bases labelled with fluorescent labels.
In each cycle, a light source 5 illuminates the functionalization surface 25, in an excitation spectral band centred on a fluorescence excitation wavelength of a fluorescent label.
Below, reference will be made to the sequence S1 captured and amplified at capture site 251. In a first cycle, a base T was hybridized.
The light source 5 generates excitation light 7, which propagates through the sample 2 to the functionalization surface 25. Under the effect of the illumination at the excitation wavelength, fluorescence light 8 is emitted by the fluorescent label C*, at a fluorescence wavelength longer than the excitation wavelength. Fluorescence photons are emitted, forming fluorescence light 8. Because of the transparency of the interface layer 23, or of the material 22 from which the electrode 21c is made, certain fluorescence photons propagate through a nanowire 30. When a fluorescence photon is absorbed at the junction 33, electron/hole pairs are formed. Under the effect of the potential difference between the first portion 31 and the second portion 32, the electrons propagate through the n-doped portion, toward the higher potential while the holes propagate in the opposite direction through the p-doped portion. This results in an increase in the electric current flowing through the detection circuit 40.
The choice of the semiconductor forming the nanowire 30 depends on the absorption spectral band of said material, said absorption spectral band needing not only to contain the fluorescence wavelength, but also preferably not to contain the excitation wavelength of the fluorescent label. The nanowire thus acts as a nanophotodetector of the fluorescence light, while not being sensitive to the excitation wavelength. Each nanowire thus forms a filter with respect to the excitation wavelength. Optionally, an absorbent layer, for example a zinc oxide layer deposited by sputtering, may be added between the functionalization surface 25 and the conductive layer 22, to reduce the spectral width of the response of the nanowires. When the semiconductor is GaAs, the fluorescent label may be Cy3 (cyanine): excitation wavelength 540 nm and emission wavelength between 555 and 600 nm.
Preferably, the diameter of the nanowires is set so as ensure sensitivity is restricted to a narrow spectral band. By narrow spectral band, what is meant is a bandwidth typically of a few tens of nm, and preferably less than 100 nm. The bandwidth corresponds to the full width at half maximum of the absorption peak in the absorption spectrum. The detection bandwidth is for example of the order of 50 nm. It is thus possible to adjust the detection spectral band so that it contains the fluorescence wavelength of the fluorescent label and so that it does not contain the excitation wavelength of the fluorescent label. The ability of nanowires to form a wavelength-selective photodetector has been described in Mokkapati et al., “Optical design of nanowire absorbers for wavelength selective photodetectors”, Sci. Rep. 2015, 5 15339. Spectral detection sensitivity may be adjusted by incorporating quantum wells or quantum dots into the junction 33. This makes it possible to obtain a junction 33 the composition of which is different from the remainder of the nanowire. The detected wavelength then corresponds to the wavelength of the gap defined by the quantum well or quantum dot.
Each nanowire thus forms a nanophotodetector. In each cycle, it is necessary not only to detect fluorescence light, but also to identify the fluorescent label that generated the fluorescence light, in order to identify the base to which it was bound. In this example, it is assumed that the nucleic bases contained in the reaction medium are labelled with a fluorescent label A*, G* and C*, respectively. It is assumed that the T bases are not labelled with a fluorescent label. In each cycle, the following is detected:
One important aspect of the invention is that discrimination is achieved by characterizing the detection electric current delivered by the nanowires.
In order to discriminate between the fluorescence light respectively emitted by each fluorescent label, each fluorescent label used has one or more emission parameters that are different from those of the others. By emission parameter, what is meant is a parameter characterizing a temporal distribution of the fluorescence intensity emitted by the fluorescent label. It may for example be a question of the maximum intensity Imax of the distribution and/or the decay time of the fluorescence light. The decay time of the fluorescence light corresponds to a time for which the fluorescence intensity decreases between two predetermined levels, for example between the maximum intensity Imax and a certain percentage of the maximum intensity, for example 50% or 80%. The decrease in fluorescence intensity may vary as
where t corresponds to time and τ is a decay constant. In this case, the decay time may be characterized by the decay constant τ.
The symbol S designates a detection signal delivered by the detection circuit 40 and formed from charge collected in each nanowire. The device comprises a processing unit 41, allowing the detection signal to be characterized, so as to identify the fluorescent label that generated the fluorescence light. The processing unit 41 may comprise a microprocessor 41, connected to a memory 42 in which instructions are stored. The processing unit forms a characterization signal S* from the detection signal.
The emission of fluorescence light allows an exploitable detection signal to be obtained in a fluorescence time of the order of one or more ns.
It may be seen that:
An absence of variation in the detection signal makes it possible to conclude that no fluorescence has been detected. This reflects hybridization of a T base to the sequence to be characterized.
The embodiment described with reference to
In a second example, shown in
When the characterization signal corresponds to an integration of the detection signal, the level reached by the characterization signal allows the fluorescent labels G*, A* and C* to be discriminated between. The same goes when the characterization signal is a mean or a median or results from another statistical indicator representative of the intensity of the detection signal during the detection period.
However, the number of sequences to be characterized immobilized on the functionalization surface, plumb with a nanowire or with a given capture site, may vary, as may the number of amplification errors. Thus, the number of identical sequences replicated per amplification at each capture site may vary from one capture site to another capture site. Now, the amplitude of the photoinduced current depends on the number of fluorescent labels labelling bases hybridized in a given cycle. To address this difficulty, a calibration sequence Sc may be grafted onto each sequence to be characterized, so as to establish the intensity levels respectively associated with each fluorescent label.
The calibration sequence Sc corresponds to a succession of bases arranged in a known order, and the location of which in the adapter is known. For example, the calibration sequence is placed in a known location in the adapter P2, which is intended to remain free after amplification. Use of a calibration sequence is particularly useful when the characterization signal corresponds to the detection signal at a given time, or comprises an integral of the detection signal.
Use of the calibration sequence makes it possible to experimentally determine the levels S*1, S*2 and S*3 at each capture site, knowing that the number of identical sequences may vary from one site to another. The levels thus established are used to interpret the detection signals detected in the cycles performed on each sequence to be characterized.
According to one variant, illustrated in
In each cycle, the processing unit 41 establishes a characterization signal S*, which characterizes the detection signal S measured across the terminals of the detection circuit 40, the detection signal resulting from an electric current formed in at least one nanowire. The characterization signal S* may comprise an integration or a derivative of the intensity measured by each nanowire taken into account. The processing unit compares the characterization to stored values or values established during the calibration period, each value being assigned to one fluorescent label.
The characterization signal makes it possible to identify, in the or each detection period, one fluorescent label among a plurality of fluorescent labels that are referred to as candidates (in this example A*, C* and G*), the characteristics of which, in terms of emission intensity or decay, are known. The characteristics may be stored in the memory 42. This allows the nucleic base hybridized in the cycle to be identified.
When three labelled bases are used, the following fluorescent labels are suited to implementation of the invention: DAPI (half-life 1.88 ns), Hoechst 33342 (half-life 1.05 ns) and Hoeschst 33258 (half-life 1.22 ns). The wavelengths at which excitation is maximum are 358 nm, 350 nm and 349 nm, respectively.
The embodiment in which the various types of complementary bases are employed simultaneously is particularly suited to implementation of the invention. The fluorescent labels bound to each base comprise a terminator, the latter being removed by cleaving following formation of the characterization signal. However, the invention may be implemented by exposing the sequences to be characterized to known bases in successive baths.
It will be noted that the fluorescent labels may be cleaved in each cycle, following formation of the characterization signal. However, this is not crucial.
It may also be useful to employ a calibration sequence when the characterization signal is a differential signal, of the time-derivative type. This may make it possible to calibrate the detection signal for the various bases. When the characterization signal is of the time-derivative type, it is preferable for the fluorescent labels to be cleaved following detection of hybridization.
Generally, use of the calibration sequence allows a response of the device at each capture site to be established. This makes it possible to take into account the fluorescence emission characteristics of each fluorescent label, the variability in the number of sequences amplified at each capture site, but also potential variability in the detection performance of each nanowire.
Preferably, the fluorescent labels are excited in the same excitation spectral band, this allowing the same light source to be used. Alternatively, the fluorescent labels are excited in different excitation spectral bands, this potentially requiring various light sources to be used.
Use of nanowires is particularly advantageous. It makes it possible to obtain an exploitable signal using a small number of sequences replicated at the same capture site. It makes it possible to reduce the size of the capture sites, relative to the prior art. It is thus possible to characterize, on a functionalization surface of given area, a larger number of sequences. The cycles may be implemented in parallel at each capture site defined on the functionalization surface. The reduction in the number of amplifications makes it possible to limit the drawbacks associated with the sequence amplification process: smaller volume of reagents required, smaller number of replicates and hence a smaller number of amplification errors.
Specifically, the detection circuit allows very low currents to be measured, of the order of a few tens of pA to a few nA. In addition, the nanowires may be closely spaced, typically with a spatial pitch of less than 500 nm (compared to 1.1-1.7 μm typically for a CMOS optical detector). This allows a device having a high ratio of working area (i.e. the area of the functionalization surface) to fluid volume to be obtained.
The excitation spectral band is preferably between 350 nm and 550 nm. The detection spectral band of each nanowire is preferably dimensioned to allow the fluorescence light to be detected while masking the excitation light. Alternatively, the detection currents, respectively formed in each nanowire during excitation and emission of fluorescence light, may be temporally separate. Advantage is taken of the fast response time (of the order of one ps) of the nanowires, as described in the publication Gallo, et al., “Picosecond response times in GaAs/AlGaAs core/shell nanowire-based photodetectors” Appl. Phys. Lett. 2011, 98 (24) 241113.
Various aspects of the design of the device according to the invention will now be described.
After heating to a temperature greater than 450° C., the islands form droplets. The gold droplets act as catalysts. The semiconductor nanowires are then formed by molecular beam epitaxy (MBE). This consists in sending one or more molecular beams toward the substrate, to achieve epitaxial growth. See
The process is not limited to use of gold as catalyst. Other catalysts may be used, for example Ga or Sn. Self-catalyzed nanowire growth is referred to when the catalyst is Ga and Ga is also a constituent of the semiconductor.
This process for example allows nanowires to be grown using As, Ga, and C and Si as p- and n-dopant, respectively, the growth temperature being 600° C.-610° C. The process continues until the nanowires reach a predetermined height. See
Following the step shown in
The conductive layer 22, then the interface layer 23, are then successively deposited on the assembly formed by the nanowires and the encapsulation layer 15. In the embodiment illustrated in
Another advantage of the bottom-up approach described with reference to
When the nanowires are distributed in clusters as described with reference to
Because of the high sensitivity of each nanowire, the number of nanowires composing a given cluster may be relatively small. Thus, the area occupied, in the plane PXY, by each cluster is small, each cluster selectively addressing a nucleotide sequence different from the other clusters. It is thus possible to provide a large number of clusters, respectively addressing various nucleotide sequences, in the same compact device.
Arrangement of the nanowires into clusters 35 may be combined with structuring the functionalization surface to form nanowells 27, as described with reference to
According to another possibility, the nanowires are obtained by etching, using a so-called top-down approach. The top-down approach is described in patent application FR2114563 filed on Dec. 27, 2021.
In the example of
An axial structure is considered to be advantageous because it favours incorporation of quantum wells or quantum dots into the nanowires, in order to adjust the absorption spectrum.
A radial structure makes it possible to provide a junction 33 extending a considerable height along the axis Z, this allowing detection sensitivity to be increased. Optionally, the radial structure shown in
Step 100: providing a sample containing nucleic acid sequences drawn from a library prepared beforehand from the DNA strand to be sequenced, which is brought into contact with the functionalization surface 25.
Step 110: capturing nucleic acid sequences by means of the functionalization surface, preferably at capture sites arranged plumb with the nanowires.
Step 115: amplifying, for example bridge amplifying, the nucleic acid sequence captured at each site. Step 115 is optional, but it allows, in each cycle, a detection signal of higher intensity to be obtained, because of the increase in fluorescence light.
Step 120: adding a reaction medium, containing nucleic bases labelled with fluorescent labels and a hybridase. Preferably, a single nucleic base is capable of being hybridized to each sequence. Each type of base is labelled with a fluorescent label called the predetermined candidate fluorescent label, which is coupled to a terminator. Bases of the same type are labelled with the same candidate fluorescent label.
Step 130: rinsing the sample.
Step 140: illuminating the functionalization surface, so as to cause fluorescence of fluorescent labels hybridized following step 120.
Step 150: detecting a detection signal across the terminals of the detection circuit.
Step 160: characterizing the detection signal, so as to identify the fluorescent label among the candidate fluorescent labels.
Step 170: cleaving the terminator, optionally cleaving the fluorescent label, and rinsing the sample.
Step 180: reiterating steps 120 to 170, until each nucleotide sequence captured at a capture site has been sequenced.
In the method described with reference to
The device takes advantage of a fast response time, typically of the order of one ns.
It will be noted that the device does not require bulky optical components to be used. In addition, the response of the device is stable, and insensitive to environmental variations: pH of the sample, temperature, presence of molecules or ions other than the biomolecule of interest. This is due to the fact that the nanowires do not make contact with the sample, but are physically and electrically isolated from the latter by the interface layer 23.
Lastly, since the device is based on nanophotodetectors, it allows a compact analysis platform to be obtained. A wafer-level manufacturing process may be used to obtain the device, this allowing cost to be lowered.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2306747 | Jun 2023 | FR | national |
