WO2021262163A1

WO2021262163A1 - Plasmonic sensors and detection

Info

Publication number: WO2021262163A1
Application number: PCT/US2020/039432
Authority: WO
Inventors: Steven Barcelo; Keith E. Moore; Raghuvir N. SENGUPTA; Fausto D'APUZZO
Original assignee: Hewlett Packard Development Co LP
Current assignee: Hewlett Packard Development Co LP
Priority date: 2020-06-24
Filing date: 2020-06-24
Publication date: 2021-12-30
Anticipated expiration: 2022-12-24

Abstract

In example implementations, an apparatus is provided. The apparatus includes a plasmonic sensor that is functionalized to immobilize a target molecule, a first light source, a second light source, and a detection device. The first light source is to provide heat to perform a polymerase chain reaction to create copies of the target molecule on the plasmonic sensor. The second light source is to induce luminescence on the plasmonic sensor. The detection device is to generate a detection signal of the target molecule based on an intensity of the luminescence that is scattered by the plasmonic sensor.

Description

PLASMONIC SENSORS AND DETECTION

BACKGROUND

[0001] Surface-enhanced luminescence techniques, such as surface- enhanced Raman spectroscopy (SERS), have emerged as leading-edge techniques for the analysis of the structure of inorganic materials and complex organic molecules. For example, in SERS, scientists engaged in the application of Raman spectroscopy have found that by producing a unique surface, upon which a molecule is later adsorbed, with a thin layer of a metal in which surface plasmons have frequencies in a range of electromagnetic radiation used to excite such a molecule and in which surface plasmons have frequencies in a range of electromagnetic radiation emitted by such a molecule, it is possible to enhance the intensity of a Raman spectrum of such a molecule.

[0002] In addition, spectroscopists utilizing spectroscopic techniques for the analysis of molecular structures have a continuing interest in improving the sensitivity of their spectroscopic techniques. Not only is improved sensitivity desirable for reducing the time of analysis, but improved sensitivity can also provide previously unachievable results. For example, improved sensitivity is directly related to lower detectability limits for previously undetected molecular constituents.

BRIEF DESCRIPTION OF THE DRAWINGS

[0003] FIG. 1 illustrates a block diagram of an example apparatus with a plasmonic sensor of the present disclosure;

[0004] FIG. 2 illustrates a block diagram of amplification and detection on an example plasmonic sensor with flexible columnar structures in a closed position; [0005] FIG. 3 illustrates a block diagram of amplification and detection on an example plasmonic sensor with flexible columnar structures in an open position; [0006] FIG. 4 illustrates an example workflow diagram for amplification and detection on a plasmonic sensor of the present disclosure;

[0007] FIG. 5 illustrates another example workflow diagram for amplification and detection on a plasmonic sensor of the present disclosure;

[0008] FIG. 6 illustrates an example workflow diagram for improved sensitivity via digestion of the present disclosure;

[0009] FIG. 7 illustrates an example of selective functionalization using a printhead of the present disclosure;

[0010] FIG. 8 illustrates an example of selective functionalization using microfluidic channels of the present disclosure; and

[0011] FIG. 9 illustrates an example flowchart for a method of thermal cycling and detecting a target molecule in a single plasmonic substrate of the present disclosure.

DETAILED DESCRIPTION

[0012] Examples described herein provide an apparatus and method to amplify and detect a target molecule in a single plasmonic sensor. As noted above, spectroscopists utilizing spectroscopic techniques for the analysis of molecular structures have a continuing interest in improving the sensitivity of their spectroscopic techniques. Not only is improved sensitivity desirable for reducing the time of analysis, but improved sensitivity can also provide previously unachievable results. For example, improved sensitivity is directly related to lower detectability limits for previously undetected molecular constituents.

[0013] The present disclosure provides a plasmonic sensor with flexible columnar structures having a metal cap. The target molecule can be bound to the metal cap on the plasmonic sensor and amplified through a polymerase chain reaction (PCR) using thermal cycling, or other types of isothermal copy procedures. With the flexible columnar structures, the target molecule may be placed in a fixed location that allows a first light source to provide more efficient localized heating at known locations during the thermal cycling. In addition,

PCR amplified target molecules fixed on the metal caps on or near “hot spot” regions may be detected via high enhancement luminescence on the substrate through a second light. The second light may be emitted at a different intensity, or power level, from the same light source as the first light that generates heat.

In another example, the apparatus may include two light sources, with one for detection and the other for localized heating.

[0014] In addition, certain modifications may be made to the primers used with the target molecule to improve sensitivity and detection. For example, the primers may replace adenine with 2-aminopurine, or a probe molecule may be added to improve a detection signal of the target molecule. Thus, the present disclosure provides an apparatus that uses a single plasmonic substrate to copy a target molecule and perform enhanced luminescence detection of the target molecule.

[0015] FIG. 1 illustrates a block diagram of an example apparatus 100 of the present disclosure. In an example, the apparatus 100 may include a plasmonic sensor 102, a first light source 104, a second light source 106, a detector 108, and a fluid delivery system 110. In an example, one or more fluids that include compounds and a target molecule 112 may be delivered via the fluid delivery system 110 over the plasmonic sensor 102. For example, the compounds and the target molecule 112 may be provided via an inlet 114, delivered over the plasmonic sensor 102, and removed via an outlet 116.

[0016] In an example, the plasmonic sensor 102 may be used to generate copies of the target molecule in a sufficient quantity to be detected by the detector 108. The plasmonic sensor 102 may be functionalized to immobilize a target molecule in known locations, as discussed in further details below. In an example, the first light source 104 may provide light to generate heat that is used to perform thermal cycling, as discussed in further details below. The thermal cycling is performed to generate copies of the target molecule. An example of the plasmonic sensor 102 is illustrated in FIG. 2, and discussed below. However, other examples of the plasmonic sensor 102 may include irregular shaped nanopillars, colloidal nanoparticles, and the like. In an example, any type of plasmonic sensor 102 that contain structures that can immobilize the target molecule may be deployed.

[0017] The second light source 106 may provide luminescence of the plasmonic sensor 102 to induce detection. The light rays or beams may be scattered off of the plasmonic sensor 102 and read by the detector 108. For example, the way the light is scattered by the target molecule may generate a signal that is detected by the detector 108. The detector 108 may convert the detected signal into an image or graph.

[0018] In an example, the detector 108 may be an optical detector or a charged coupled device (CCD) or spectrometer to collect spectral images of the plasmonic sensor 102. The detector 108 may be an imaging system, a multi channel spectrophotometer, or any number of other optical sensors.

[0019] In an example, the detector 108 may include lenses, filters, diffraction gratings, and other devices (not shown) to focus the incoming light scattered by the plasmonic sensor 102 onto a detector array. In an example, the detector 108 may divide the incoming light into different channels, each of which are sent to a different sensor within the detector 108, providing multi-spectral analysis of the light scattered by the plasmonic sensor 102. The detector 108 may perform brightfield, dark-field, fluorescence, hyperspectral, and other optical analyses. Thus, the apparatus 100 provides a device that can generate copies of a target molecule and perform detection within a single plasmonic substrate.

[0020] In an example, the first light source 104 and the second light source 106 may be laser light sources. Although two different light sources 104 and 106 are illustrated in FIG. 1 , it should be noted that the first light source 104 and the second light source 106 may be deployed as a single light source or single device that emits light at different wavelengths or of different intensities.

[0021] In an example, the first light source 104 and the second light source 106 may emit light at similar wavelengths, but different intensities or power levels. In an example, the first light source 104 and the second light source 106 may emit light at different wavelengths at the same intensity or power level. In an example, the first light source 104 and the second light source 106 may emit light at different wavelengths and different intensities or power levels. [0022] In an example, the first light source 104 may emit light to generate heat in localized areas on the plasmonic sensor 102. The light from the first light source 104 may be within a near ultraviolet (UV), visible, or near infrared (IR) wavelength range. For example, the wavelength may be approximately 300 nanometers (nm) to 3000 nm. In an example, the light from the first light source 104 may be emitted at an intensity of approximately 0.1 to 10 kilowatts per square centimeter (kW/cm²).

[0023] In an example, the second light source 106 may emit light to provide luminescence over the plasmonic sensor 102 to induce detection of the target molecule. The light from the second light source may be near UV, visible, or near IR. For example, the wavelength may be approximately 500 to 900 nm. In an example, the light from the second light source 106 may be emitted at an intensity of approximately 1 to 1000 Watts per square centimeter (W/cm²).

[0024] In an example, the target molecule is a nucleic acid (e.g., deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) that may be a constituent of any compound, composition of matter, chemical, biological cell, bio-matter, and the like, that is to be detected by the detector 108. The compounds may include various chemicals, primers, enzymes, and the like, that are provided to promote a polymerase chain reaction (PCR) to generate copies of the target molecule with the help of thermal cycling with heat generated from the light emitted by the first light source 104. The compounds and target molecule 112 may be provided in a series of different fluids that are delivered to the plasmonic sensor 102 via the fluid delivery system 110.

[0025] The plasmonic sensor 102 may include a variety of different types of surface enhanced luminescence sensors. For example, the plasmonic sensor 102 may include a surface enhanced Raman spectroscopy (SERS) sensor, a surface enhanced infrared absorption (SEIRA) sensor, a surface enhanced fluorescence (SEF) sensor, and the like.

[0026] FIG. 2 illustrates an example of amplification and detection on the plasmonic sensor 102 of the present disclosure having flexible columnar structures in a closed position. In an example, the surface of the plasmonic sensor 102 of the present disclosure may include a plurality of flexible columnar structures 202i and 2022 (hereinafter collectively referred to as flexible columnar structures 202 or individually referred to as a flexible columnar structure 202) supported on a substrate (e.g., a silicon, glass, quartz, silicon nitride, and the like). Although two flexible columnar structures 202 are illustrated in FIG. 2, it should be noted that there may be millions of flexible columnar structures 202 deployed on the surface of the plasmonic sensor 102.

[0027] In an example, the flexible columnar structures 202i and 202₂ may each have a metal cap 204i and 204₂, respectively (hereinafter collectively referred to as metal caps 204 or individually referred to as metal cap 204). The metal caps 204 may be made of any conductive metal, such as for example, silver, gold, aluminum, copper, and the like.

[0028] In an example, the flexible columnar structures 202 and the metal cap 204 may have a mushroom shape. However, it should be noted that the flexible columnar structures 202 with the metal caps 204 may include other shapes that are within the scope of the present disclosure. In an example, the flexible columnar structure 202 may include shapes such as a nanocones, nanopyramids, nanorods, nanobars, nanopoles, nanograss, and the like.

[0029] In an example, the flexible columnar structures 202 may have nano dimensions that are as small as a few tens of nanometers (nm) in height and a few nanometers in diameter or width. For example, the flexible columnar structures 202 may have a diameter of approximately 50 nm to 500 nm, a height of approximately 50 nm to 2 microns, and a gap between the flexible columnar structures 202 of approximately 20 nm to 500 nm.

[0030] In an example, the metal caps 204 may have a diameter on the order of approximately 20 nm to 500 nm. The metal caps 204 may be substantially spherical, prolate spheroidal, oblate spheroidal, disk-like, and the like.

[0031] In an example, the flexible columnar structures 202 may be spaced such that certain groups of the flexible columnar structures 202 are bent towards one another. In an example, the flexible columnar structures 202 may bend towards each other under action of microcapillary forces induced by removal of a fluid carrier or liquid carrying the compounds and target molecule. [0032] In an example, a strong enhancement in surface-enhanced luminescence may be obtained by the flexible columnar structures 202 when they are collapsed into groups. The enhancement may be based on intense local electric fields generated by the plasmon resonance of adjacent metal caps 204 at the top of the bent flexible columnar structures 202.

[0033] As noted above, FIG. 2 illustrates an example of amplification with the flexible columnar structures 202 in a closed position. Said another way the flexible columnar structure 202i may be bent towards an adjacent flexible columnar structure 202₂ such that the metal cap 204i may touch or be close to the metal cap 204₂. In some examples, where more than two flexible columnar structures 202 are grouped together, the flexible columnar structures 202 may be bent to form other two-dimensional shapes (e.g., when viewed from above). For example, three flexible columnar structures 202 may be bent towards one another to form a triangle or pyramid, four flexible columnar structures 202 may be bent towards one another to form a square or rhombus, and so forth.

[0034] In an example, a forward primer 208, a target molecule 210, a reverse primer 212, and an enzyme 214 may be added to the plasmonic sensor 102. In an example, the forward primer 208 may be modified to include thiol groups to attach to the surfaces of the metal caps 204 via a covalent bond. In an example, the plasmonic sensor 102 may be coated with a molecule with carboxyl terminal groups, such as carboxylic acid, which in the presence of polyethylene glycol and salts (e.g., Mg²⁺) will bind to primers without further modification. In an example, the plasmonic sensor 102 may be coated with a molecule with carboxyl terminal groups, such as carboxylic acid, which in the presence of polyethylene glycol and salts (e.g., Mg²⁺) will bind to the target molecules without further modification. The forward primer 208 may provide sites for annealing to the target molecule 210. Examples of the forward primer 208 may include 2-aminopurine substituted for adenine, as well as thiolated primer sequences.

[0035] In an example, the target molecule 210 may be a nucleic acid that may be a constituent of any compound, composition of matter, chemical, biological cell, bio-matter, and the like that is to be detected by the apparatus 100 via the plasmonic sensor 102. In an example, the target molecule 210 may be DNA or RNA.

[0036] In an example, the reverse primer 212 may have a similar composition as the forward primer 208. For example, the reverse primer 212 may include 2-aminopurine substituted for adenine, thiolated primer sequences, or a Raman active probe molecule at one end of the primer. However, the reverse primer 212 may replicate in an opposite direction as the direction of replication for the forward primer 208. In some examples, the reverse primer 212 can be marked with a probe molecule for detection. The probe molecule may be a resonant Raman molecule or dye. In an example, the probe molecule may be rhodamine 6G, fluorescein, or acridine orange.

[0037] In an example, the enzymes 214 may promote the PCR process. The enzymes may include polymerase, nucleoside triphosphate (NTP) building blocks to extend the forward primer 208 with the target molecule 210.

[0038] Heat may be applied to the plasmonic sensor 102 to perform thermal cycling. The thermal cycling may create copies 206i to 206_n (hereinafter also referred to collectively as copies 206 or individually as a copy 206) of the target molecule 210. After thermal cycling for a pre-determined number of cycles to generate a sufficient number of copies 206 of the target molecule 210, the plasmonic sensor may be exposed to light or luminescence. The number of cycles may be a function of an amount of heat generated by the first light source 104, a replication rate of the target molecule 210, a signal strength created by the plasmonic sensor 102, and the like. In an example, 20-40 thermal cycles may be performed when the target molecule is DNA. In another example, the plasmonic sensor 102 can be tested for the detection of the target molecule 210 after each thermal cycle. While the signature of the target molecule 210 may be detected from the first cycle, cycling is continued and the signal is recorded until the last cycle is performed or the signal intensity and evolution is sufficient to confidently interpret the results. The luminescence may allow the detector 108 to detect the presence of the copies 206 of the target molecule 210.

[0039] FIG. 3 illustrates a block diagram of amplification and detection on the plasmonic sensor 102 with the flexible columnar structures 202 in an open position. For example, in FIG. 3, the flexible columnar structures 202i and 202₂ may be relatively straight or parallel to one another. When the flexible columnar structures 202i and 202₂ are straight, a gap 220 of more than 10 nm may be formed between the metal caps 204i and 204₂.

[0040] The open position may have some advantages for thermal cycling compared to the closed position illustrated in FIG. 2. For example, in the open position, hot spots typically present in metallic nanoparticles with gaps less than 10 nm are not formed during thermal cycling. This may reduce risk of “burning” molecules already in hot spots, as well as reduce temperature gradients and allow a more uniform heating of the metal surface of nanoparticles.

Furthermore, the open position may allow a further separation of the wavelength used for heating compared to the one used for detection. The open position may also provide better sensitivity due to better trapping of the target molecules 210 in hot spots once they are formed by collapsing the structure into the closed position illustrated in FIG. 2.

[0041] The forward primers 208, target molecule 210, the reverse primers 212, and the enzymes 214 may be delivered to the plasmonic sensor 102, as described above in FIG. 2. Heat may be applied to perform thermal cycling and create copies 206 of the target molecule 210. The heat may be modulated by varying the light source intensity to achieve multiple stable temperatures. After a predefined number of thermal cycles, a light source may provide luminescence to perform detection of the copies 206 of the target molecule 210 using the detector 108.

[0042] FIG. 4 illustrates an example workflow diagram 400 for amplification and detection on the plasmonic sensor 102 of the present disclosure. FIG. 4 illustrates an example in which 2-aminopurine may replace adenine in the forward primers 208. The adenine is re-introduced via the NTP building blocks as the reverse primer 212 is replicated, as discussed in further details below. The detection of adenine, present within the target molecule, may provide a luminescent signal that can be detected by the detector 108 indicating the presence of copies 408 of the target molecule 402.

[0043] The workflow diagram 400 begins at block 402. At block 402, forward primers 208 are delivered to the plasmonic sensor 102. In an example, the metal caps 204 on the flexible columnar structures 202 may be functionalized with a compound to allow the forward primers 208 to be covalently bonded to the metal caps 204. In an example, 5’-SH-(CH₂)₆ modified primers may be attached to the metal caps 204. The forward primers 208 may then be attached to the metal caps 204 via the 5’-SH primers and immobilized. In an example, the forward primers 208 may substitute adenine with 2-aminopurine.

[0044] At block 404, a target molecule 210 may be delivered to the plasmonic sensor 102. The target molecule 210 may attach to one of the forward primers 208 and anneal with the immobilized forward primer 208.

[0045] At block 406, an enzyme may be delivered to the plasmonic sensor 102 to allow the forward primer 208 to be extended with the attached target molecule 210 (e.g., DNA) to create a copy 418 of the target molecule 210. In an example, the enzyme may include a polymerase along with NTP building blocks. The NTP building blocks may include adenine. Thus, the copy 418 of the target molecule 210 may include adenine that can generate a signal under luminescence that can be detected. The signal generated by adenine incorporated into the target molecule 210 may be sufficiently distinct from the adenine present in ATP to positively determine the presence of the target molecule 210.

[0046] At block 408, heat may be generated locally over various locations of the plasmonic sensor 102. The heat may be generated locally by directing light from a first light source (e.g., the first light source 104) at desired locations where the forward primers 208 are known to be located. The heat may separate the target molecule 210 from the copy 418.

[0047] At block 410, a reverse primer 212 may be delivered to the plasmonic sensor 102. The reverse primer 212 may also be made with 2-aminopurine.

The plasmonic sensor 102 may be allowed to cool. In an example, the fluid around small areas that are locally heated may provide passive cooling for the plasmonic sensor 102. For example, when the irradiation from the first light source stops, the rest of the fluid may act as a heat sink for the fluid layer at the metal caps 204 that were locally heated. For example, the lower temperature of the fluid or liquid used to deliver the reverse primer 212 may be used to cool the plasmonic sensor 102. Cooling the plasmonic sensor 102 may allow the target molecule 210 to attach to another immobilized forward primer 208. In addition, the reverse primer 212 may attach to the copy 418.

[0048] At block 412, enzymes that were provided in block 406 may be provided again to promote extension of the forward primer 208 and the reverse primer 212. Thus, two additional copies 418 of the target molecule 210 may be generated. The copy 418 generated from the reverse primer 212 may also include adenine as the reverse primer 212 is extended from the copy 418 of the target molecule 210.

[0049] In an example, blocks 408, 410, and 412 may be referred to as a thermal cycle. The blocks 408, 410, and 412 may be repeated over a predetermined number of times to perform multiple thermal cycles. Each thermal cycle may be performed to generate additional copies 418 of the target molecule 210. The thermal cycle may be repeated until enough copies 418 of the target molecule 210 are generated to be detected by a detector (e.g., the detector 108).

[0050] After the desired number of thermal cycles are completed, the workflow diagram 400 proceeds to block 414. At block 414, a sufficient number of copies 418 of the target molecule 210 are generated. A light may be directed to the plasmonic sensor 102 for luminescence detection of the target molecule 210.

[0051] FIG. 5 illustrates an example workflow diagram 500 for amplification and detection on the plasmonic sensor 102 of the present disclosure. FIG. 5 illustrates an example in which probes may be used to amplify a detection signal of the target molecule 210.

[0052] The workflow diagram 500 begins at block 502. At block 502, forward primers 208 are delivered to the plasmonic sensor 102. In an example, the metal caps 204 on the flexible columnar structures 202 may be functionalized with a compound to allow the forward primers 208 to be covalently bonded to the metal caps 204. In an example, 5’-SH-(CH₂)₆ modified primers may be attached to the metal caps 204. The forward primers 208 may then be attached to the metal caps 204 via the 5’-SH primers and immobilized. [0053] At block 504, a target molecule 210 may be delivered to the plasmonic sensor 102. The target molecule 210 may attach to one of the forward primers 208 and anneal with the immobilized forward primer 208.

[0054] At block 506, an enzyme may be delivered to the plasmonic sensor 102 to allow the forward primer 208 to be extended with the attached target molecule 210 (e.g., DNA) to create a copy 518 of the target molecule 210. In an example, the enzyme may include a polymerase along with NTP building blocks.

[0055] At block 508, heat may be generated locally over various locations of the plasmonic sensor 102. The heat may be generated locally by directing light from a first light source (e.g., the first light source 104) at desired locations where the forward primers 208 are known to be located. The heat may separate the target molecule 210 from the copy 518.

[0056] At block 510, a reverse primer 212 may be delivered to the plasmonic sensor 102. The reverse primer 212 may be labelled with a probe 520. The probe 520 may be a resonant Raman molecule or dye that can be easily detected via luminescence by a detector (e.g., the detector 108 in FIG. 1). The plasmonic sensor 102 may be allowed to cool. For example, the lower temperature of the fluid or liquid used to deliver the reverse primer 212 may provide passive cooling to the plasmonic sensor 102. The cooling can also be provided by keeping the plasmonic sensor 102 at a constant temperature with a thermostat. Cooling the plasmonic sensor 102 may allow the target molecule 210 to attach to another immobilized forward primer 208. In addition, the reverse primer 212 may attach to the copy 518.

[0057] At block 512, enzymes that were provided in block 506 may be provided again to promote extension of the forward primer 208 and the reverse primer 212. Thus, two additional copies 518 of the target molecule 210 may be generated.

[0058] In an example, blocks 508, 510, and 512 may be referred to as a thermal cycle. The blocks 508, 510, and 512 may be repeated over a predetermined number of times to perform multiple thermal cycles. Each thermal cycle may be performed to generate additional copies 518 of the target molecule 210. The thermal cycle may be repeated until enough copies 518 of the target molecule 210 are generated to be detected by a detector (e.g., the detector 108).

[0059] After the desired number of thermal cycles are completed, the workflow diagram 500 proceeds to block 514. At block 514, a sufficient number of copies 518 of the target molecule 210 are generated. The copies 518 may be labelled with the probe molecule 520. A light may be directed to the plasmonic sensor 102 for luminescence detection of the target molecule 210. [0060] It should be noted that FIGs. 4 and 5 illustrate an example of how the target molecule 210 can be added to the forward primer 208 to make copies of the target molecule 210. However, it should be noted that the plasmonic sensor 102 may be functionalized to immobilize the target molecule 210 to the metal caps 204 and then add the forward primers 208.

[0061] As noted above, the plasmonic sensor 102 may be coated with a molecule with carboxyl terminal groups, such as carboxylic acid, which in the presence of polyethylene glycol and salts (e.g., Mg²⁺) will bind to the target molecules 210 without further modification. The forward primers 208 may then be added. The forward primers 208 may attach to the target molecule 210. Enzymes may then be delivered to allow the forward primer 208 to be extended with the attached target molecule 210. Thermal cycling may then be performed to generate copies of the target molecule, as described above and illustrated in FIGs. 4 and 5.

[0062] FIG. 6 illustrates an example workflow diagram 600 for improved sensitivity via digestion of the present disclosure. In some examples, the workflow diagram 600 can be performed after the PCR is performed to replicate the target molecule 210. In an example, the workflow diagram 600 can be performed after the workflow diagram 500.

[0063] At block 602, the copies 518 may be replicated on the metal caps 204 of the flexible columnar structures 202. The copies 518 may be labelled with a probe molecule 520.

[0064] At block 604, the plasmonic sensor 102 may be rinsed. The plasmonic sensor 102 may be rinsed with a solvent, for example, deionized water, ethanol, a TE buffer (e.g., a buffer used in procedures involving nucleic acids that comprise 10 millimolars (mM) Tris and 1 mM ethylenediaminetetraacetic acid (EDTA) with the pH of the solution adjusted to 8), and the like to remove interfering compounds in the master mix. For example, compounds such as the NTP building blocks or remaining polymerase may be removed.

[0065] At block 606, heat may be generated again by applying a light from the first light source 104 towards the locations with the copies 518 of the target molecule 210. The heat may separate the double stranded copies 518 of the target molecule 210 for easier digestion.

[0066] In an example, a deoxyribonuclease (DNase) may be delivered to the plasmonic sensor 102. The DNase may be an enzyme that catalyzes the hydrolysis of the target molecule 210 (e.g., DNA) into oligonulceotides and smaller molecules. For example, the DNase may fragment the copies 518 into shorter strands, a subset of which contain the probe 520.

[0067] At block 608, the copies 518 of the target molecule 210 may be digested that release the probe molecule 520 into solution. The probe molecules 520 may have more direct interaction with plasmonic sensing hot spots on the plasmonic sensor 102.

[0068] FIGs. 7 and 8 illustrate different examples of how selected locations of the plasmonic sensor 102 may be functionalized. FIG. 7 illustrates an example that uses a printhead 704. In an example, a stage 702 may hold the plasmonic sensor 102. The stage 702 may be movable along a three dimensional axis (e.g., an x-y-z axis).

[0069] A processor 708 may be communicatively coupled to a printhead 704 and the stage 702. The processor 708 may control dispensing of a fluid via the printhead 704 and control movement of the stage 702. A reservoir 706 may be coupled to the printhead 704. The reservoir 706 may include a fluid that is used to functionalize the plasmonic sensor 102 (e.g., via thiolated primer sequences that attach to the metal caps 204).

[0070] In an example, the printhead 704 may be a thermal inkjet (TIJ) resistor. The TIJ resistor may locally heat a fluid to generate a bubble. The energy released by the bubble may cause a drop of fluid to be ejected from the printhead 704.

[0071] In an example, the processor 708 may control the printhead 704 and the stage 702 such that fluid from the reservoir 706 is dispensed onto desired locations 710i to 710_m (hereinafter collectively referred as locations 710 or individually referred to as a location 710) of the plasmonic sensor 102. As a result, the locations 710 may be known to be where the target molecules 210 are replicated. This may help to improve the accuracy of where the light emitted from the first light source 104 and the second light source 106 are directed. As a result, heat may be generated more precisely and efficiently for the thermal cycling and light may be more precisely directed at the target molecules 210 for detection via luminescence.

[0072] In an example, the printhead 704 may be connected to a plurality of different reservoirs 706 that contain different primers to detect different target molecules. In an example, a plurality of different printheads 704 connected to respective reservoirs 706 with different primers may be deployed. As a result, different primers may be dispensed on desired locations 710 to detect different target molecules. For example, a first primer may functionalize the location 710i to detect DNA. A second primer may functionalize the location 710₂ to detect RNA, and so forth. Thus, selective functionalization may enable multiplexing to detect different target molecules on a single plasmonic substrate. [0073] FIG. 8 illustrates an example of selective functionalization using microfluidic channels 804i to 804_o (hereinafter referred to individually as a channel 804 or collectively as channels 804). In an example, the microfluidic channels 804 may include an inlet 802 and respective outlets 8O6₁ to 806_o. Although a single inlet 802 is illustrated, it should be noted each channel 804 may have a respective inlet 802. In other words, a plurality of inlets 802 may also be deployed.

[0074] Each channel 804 may run over a different portion of the plasmonic sensor 102. Thus, the locations of the target molecule 210 to be detected may be known to be the locations adjacent to the channels 804. In an example, each channel 804 may deliver a different primer. As a result, different locations on the plasmonic sensor 102 may be functionalized with different primers to detect different target molecules. In other words, the channels 804 may also be used to enable multiplexing to detect different target molecules on a single plasmonic substrate.

[0075] FIG. 9 illustrates a flowchart for a method 900 for thermal cycling and detecting a target molecule in a single plasmonic substrate of the present disclosure. In an example, the method 900 may be performed by the apparatus 100 illustrated in FIG. 1 , and described above.

[0076] At block 902, the method 900 begins. At block 904, the method 900 functionalizes a surface of flexible columnar structures having a metal cap with a forward primer. In an example, the forward primer may be attached to 5’-SH modified primers that are attached to metal caps of a flexible columnar structure on a plasmonic sensor. The forward primer may be covalently bonded to the 5’- SH compounds. In an example, the forward primer may substitute adenine with 2-aminopurine.

[0077] At block 906, the method 900 adds a target molecule to anneal with the forward primer. In an example, the target molecule may be DNA. However, the target molecule may be a constituent of a larger biomass or cell which is preprocessed to release DNA, or RNA, that is to be detected by the plasmonic sensor.

[0078] At block 908, the method 900 adds an enzyme to create a copy of the target molecule via the forward primer. The enzyme may be polymerase with NTP building blocks. The enzyme may allow the forward primer to extend with the strand of the target molecule.

[0079] At block 910, the method 900 thermal cycles the target molecule with a first light source to create copies of the target molecule on the surface of the flexible columnar structures having the metal cap. In an example, the thermal cycle may include applying heat to separate the target molecule from the copy of the target molecule. The heat may be generated by directing light from a first light source towards the plasmonic sensor.

[0080] In an example, the light may be directed at particular locations on the plasmonic sensor. As discussed above, a printhead or microfluidic channels may be used to deliver precise amounts of the forward primer to select locations on the plasmonic sensor. Thus, the light may be directed at the same locations where the forward primer was dispensed.

[0081] A reverse primer may be added and the plasmonic sensor may be allowed to cool. The reverse primer may attach to the copy of the target molecule created by the forward primer. The target molecule may attach to another forward primer on the metal caps of the flexible columnar structure.

The enzymes that were delivered in the block 908 may encourage extension of the reverse primer and the forward primer to create a second and third copy of the target molecule.

[0082] In an example, the thermal cycle may be repeated a predefined number of times. The number of times the thermal cycle is repeated may be a function of an amount of heat that is generated, a replication rate of the target molecule, and a signal strength to detect the target molecule, as well as the concentration of the enzyme, target molecule, and NTP building blocks.

[0083] At block 912, the method 900 detects the target molecule via luminescence induced by a second light source. For example, a second light source emit a second light towards the plasmonic sensor to provide luminescence. The luminescence may provide light signals that are scattered by the plasmonic sensor towards a detector. The detector may then generate detection signals that can be correlated to the light signal that the target molecule is known to generate.

[0084] In an example, the light from the first light source and the second light source may be the same wavelength, but different intensity or power levels. In an example, the light from the first light source and the second light source may be different wavelengths, but the same intensity or power level. In an example, the light from the first light source and the second light source may be different wavelengths and different intensity or power levels. At block 914, the method 900 ends.

[0085] It will be appreciated that variants of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.

Claims

1. An apparatus, comprising: a plasmonic sensor functionalized to immobilize a target molecule; a first light source to provide heat to perform a polymerase chain reaction to create copies of the target molecule on the plasmonic sensor; a second light source to induce luminescence on the plasmonic sensor; and a detection device to generate a detection signal of the target molecule based on an intensity of the luminescence that is scattered by the plasmonic sensor.

2. The apparatus of claim 1 , further comprising: a fluid delivery system to deliver compounds to create a polymerase chain reaction and deliver the target molecule to the plasmonic sensor.

3. The apparatus of claim 2, wherein the fluid delivery system comprises a printhead to eject a primer on select locations of the plasmonic sensor.

4. The apparatus of claim 3, wherein a different primer is selected on different locations of the plasmonic sensor to detect different target molecules.

5. The apparatus of claim 2, wherein the fluid delivery system comprises a plurality of microfluidic channels to deliver a primer on selection locations of the plasmonic sensor.

6. The apparatus of claim 5, wherein a different primer is delivered in each microfluidic channel of the plurality of channels to detect different target molecules.

7. The apparatus of claim 1 , wherein the first light source and the second light source are generated at different power levels or different wavelengths by a single device.

8. A method, comprising: functionalizing a surface of flexible columnar structures having respective metal caps with a first forward primer; adding a target molecule to anneal with the first forward primer; adding an enzyme to create a first copy of the target molecule via the first forward primer; thermal cycling the target molecule with a first light source to create a second copy and a third copy of the target molecule on the surface of the flexible columnar structures having the respective metal caps; and detecting the target molecule via luminescence induced by a second light source.

9. The method of claim 8, wherein the thermal cycling is performed with the flexible columnar structures in an open position.

10. The method of claim 8, wherein the thermal cycling is performed with the flexible columnar structures in a closed position.

11. The method of claim 8, wherein the thermal cycling comprises: applying heat to separate the target molecule from the first copy of the target molecule; adding a reverse primer; and allowing a plasmonic sensor to cool such that the reverse primer attaches to the first copy of the target molecule and the target molecule attaches to a second forward primer, wherein the reverse primer forms the second copy of the target molecule and the second forward primer forms the third copy of the target molecule.

12. The method of claim 8, wherein the target molecule comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and the method further comprises: rinsing a plasmonic sensor after the thermal cycling; applying heat to separate double stranded DNA or RNA; adding an enzyme to digest the DNA or RNA to release a probe molecule into a solution to increase a sensitivity of the detecting.

13. An apparatus, comprising: a plasmonic sensor with a plurality of flexible columnar structures with respective metal caps; a fluid delivery system to deliver a forward primer containing 2- aminopurine, a target deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and a polymerase enzyme to the plasmonic sensor; a first laser to heat the target DNA or RNA to promote thermal cycling which allows for a polymerase chain reaction to generate copies of the target DNA or RNA that include adenine; a second laser to provide luminescence on the plasmonic sensor; and an optical sensor to detect the target DNA or RNA based on light of the luminescence that is scattered by the plasmonic sensor and the adenine in the copies of the target DNA or RNA.

14. The apparatus of claim 13, wherein the first laser emits a first light having a wavelength of between 300 to 3000 nanometers (nm) and the second laser emits a second light having a wavelength of between 500 to 900 nm.

15. The apparatus of claim 13, wherein the first laser emits light at an intensity of between 0.1-10 kilowatts per square centimeter (kW/cm²) and the second laser emits light at an intensity of 1 to 1000 Watts per square centimeter (W/cm²).