CROSS-REFERENCES TO RELATED APPLICATIONS

[00011 This application claims the benefit of United States Provisional Patent Application Number 62/783,049, filed on December 21, 2018, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE IN VENTION

[00021 Reaction vessels are ofte used to perform various operations on DNA strands that can include operations such as polymerase chain reaction (FOR) and DNA sequencing. Polymerase chain reaction (PG R) has become an essential technique in the fields of life sciences, clinical laboratories, agricultural science, environmental science, and forensic science. PCR requires thermal cycling or repeated temperature changes between two or three discrete temperatures, to amplify specific nucleic acid target sequences. To achieve such thermal cycling, conventional bench-top thermal cyclers generally use a metal heating block powere by Peltier elements. Unfortunately, this metho of thermally cycling the materials within the reaction vessels cart be slower than desired. For these reasons, alternate means that improve the speed and/or reliability of the thermal cycling are desirable.

SUMMARY OF THE INVENTION

[00031 This disclosure relates to methods and apparatus suitable for use with a reaction vessel.

[00041 In some embodiments, a reaction vessel assembly includes the following: a reaction vessel, including; a housing component a reaction chamber defined by the housing component; and a light absorbing layer conforming to a portion ©fan interior- facing surface of the housing component that defines the reaction chamber, th light absorbing layer comprising an electrically conductive pathway; a first energy source (e.g., a light source such as an LED) configured to direct light through at least a portion of the housing component at a portion of the electricall conductive pathway; and a second energy source (e.g., an electrical source such as a DC source, an AC source, a battery, etc.) configured to direct electrical energy through the electrically conductive pathway.

[0005| In some embodiments, the reaction vessel assembly also includes a processor configured ¹ to determine a temperature within die reaction chamber based upon a voltage change (e,g., with a measured voltage drop) m the electrical energy afte passing throug the electrically conductive pathway. In some embodiments, the electrical energy is conducted through an: entirety of the light absorbing layer, hr Other embodiments, thelight absorbing: layer includes a first layer in direct contact with the housing component and a second layer stacke ato the first layer that forms the electrically conductive pathway, wherein the first layer is electrically insulated from the second layer.

[00061 in some embodiments, the reaction vessel assembly includes a processor configured to determine a first temperature within the reaction chamber based upon a voltage change in the electrical energy after passing through the first electrically conductive pathway. In some embodiments, the electrical energy is directed through an entirety of the light absorbing layer.

[00071 M some embodiments, the processor is further configured to: compare the first temperature to a desired temperature; and in response to a result of said comparison, cause the light source to adjust an amount of fight directed at the portion of the light absorbing layer. For example, the processor may determine that the first temperature is equal to or greater than the desired temperature. In response, the processor may cause the light source to sto d irecting li ght at the portion of the light absorbing layer. Alternatively, the processor may decrease an amount of light directed at the portion of the light absorbing layer (e _> g _? by decreasing power supplied to the light source). As another example, the processor may determine that the first temperature is less than the desired temperature, an in response may increase an amount of light directed at the portion of the light absorbing layer,

[0008] In some embodiments, the light absorbing layer comprises a first layer in direct contact with the housing component and a second layer stacked atop the first layer that forms the first electrically conductive pathway, wherein the first layer is electrically insulated from the second layer. In some embodiments, the first layer is electrically noii-conductive.

[00O9 j In some embodiments, the reaction vessel assembly includes: a second electrically conductive pathway separate and distinct from the first electrically conductive pathway. I some embodiments, the first electrically conductive pathway covers a first portion of the housing component and the second electrically conductive pathway covers a second portion of the ousing component, and wherein the reaction vessel assembly further comprises a processor configured to determine a temperature of the first and second portions of the housing component based oh respective measure voltage changes in electrical energy after 5 passing through the first and second electrically conductive pathways.

[0010! in some embodiments, the First electrically conductive pathway has a serpentine geometry in which adjacent segments of the first electrically conductive pathway are separated by a ga less than half as wide as a width of each of the adjacent segments of the first electrically conducti ve pathway.

If} [00111 1» some embodiments, the: light absorbing layer is disposed along the surface of the housing component at a variable density. In some embodiments, the light absorbing layer is disposed at a relatively hig density along a peripheral portion of the surface of the housing component, and at a relatively low density along a central portion of the surface of the housing component in some embodiments, the light absorbing layer is disposed at a 15 relatively low density along a peripheral portion of the surface: of the housing component, and at a relatively high density along a central portion of the surface of the housing _: component. I some embodiments, the light absorbing layer comprises two or more discrete regions,

10012! in some embodiments, a method of operating a reaction vessel assembly including a reaction vessel with a reaction chamber defined by a housing component ay include 0 directing ! ight from a light source throug at least a portion of the housing component at a portion of the light absorbing layer to cause the light absorbing layer to convert the light into heat energy, wherein the l ght absorbing layer comprises a first electrically conductive pathway; directing, by an electrical energy source, electrical energy through t ¾ first electrically conductive pathway; measuring a voltage change across the first electrically 5 conductive pathway; and determining a first temperature within the reaction chamber based upon a voltage change in the electrical energy after passing through the first electrically conductive pathway.

[00131 fc some embodiments _* th electrical energy is directed through an entirety of the light absorbing layer. In some embodiments, the light absorbing layer is disposed along the 30 surface of the housing component at a variable density. In some embodiments, the light absorbing layer is disposed at a relati vely high density along a peripheral portion of the surface of the housing component, and at a relatively low density along a central portion of tile surface of the housing component. In some embodiments, the light absorbing layer is disposed at a relati vely low densi ty along a peripheral portion of the surface of the housing component, and at a relati vel y high density along a central portion of the surface of the housing component.

5 [00141 In some e bodi ments, the method includes comparing the first temperature to a desired temperature ; and i n response to a resu lt o f said comparison, causing t he light source to adjust an amount of light directed at the portion of the light absorbing layer. For example, the processor may determine that the first temperature is equal to or greater than the desired temperature. I response, the processor may cause th light source to stop directing light at:0 the portion of the light absorbing layer. Alternatively, the processor may decrease an amount of light directed at the portion of the light absorbing layer (e.g., b decreasing power supplied to the light source). As another example, the processor may determine that the first temperature is less than the desired temperature, and in response may increase an amount of light directed at th e portion of the light absorbing layer. 5 [OOlS j In some embodiments, a reaction vessel assembly includes a reaction vessel,

wherein the reaction vessel includes: a reaction chamber; and an energy absorbing layer disposed along the reaction chamber, the energy absorbing layer comprising a first electrically conductive pathway. The reaction vessel assembly may also include an energy source confi gured to direct energy at a portio of the energy absorbing l ay er; an electrical0 energy «Ource configured to direct electrical energy through the first electrically conductive pathway; and a processor. The processor may be configured to: determine a first temperature within the reaction: chamber based upon a voltage change of the electrical energy after passing through the first electrically conductive pathway: determine that the first temperature is equal to or greater than a desired temperature; and in response to said determination, cause5 the en ergy source to stop directing energ at the portion of the en ergy·' absorbing layer.

[00161 Other aspects and advantages of the invention will become apparent from the following detailed description taken in conjunction with the accompanying drawings which illustrate, by way of example, the principles of the described embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

100171 The disclosure will be readily understood by the following detaile description in conjunction with the accompanying drawings, wJberem like reference numerals designate like structural elements, and in which: [0018} FIG. 1 A shows an exemplary reaction vessel suitable for use with the described embodiments;

[00191 FIG. IB shows another exemplary reaction vessel suitable for use with the describe embodiments;

{00201 FIG, 1C show's how air gap regions establish robust barriers that reduce the lateral transfer of heat between adjacent reaction vessels;

[00211 FIG. 2 shows a schematic cross-sectional side view of a reaction vessel and how reaction chamber can be closed;

[00221 FIG. 3 A show's a cross-sectional side view' of a reaction vessel arid how a light absorbing layer can be separated into: discrete regions; [00231 FIGS. 3B -3C show' cross-sectional side view's of a reaction vessel being illuminated by an array of energy sources;

[00241 FIG, 3 D show's a cross-sectional side v ew' of a th ermal profile of a portion of a reaction vessel with different size gaps between discrete regions of a light absorbing layer;

[0025] FIG. 4A show's a schematic perspective view' of a reaction vessel having a reaction chamber taking the form of a serpentine channel;

[00261 FIG. 4B shows a top view of the reaction vessel depicted in FIG, 4A with labels indicating each of discrete regions 406-1 throug 406-8 of a light absorbing layer;

[00271 FIG. 4C show's another embodiment of a reaction vessel that includes a light absorbing layer with conformal discrete regions; [0028] FIG. 5 A shows a reaction vessel that includes a housing component having a light absorbing layer made up of two discrete regions;

[00291 FIG 5B shows how an energy source can be offset toward one of the two discrete regions such that one of the discrete regions receives more energy from the energy source than the other discrete region; [ O30J FIG. 5C shows a top view of the reaction vessel depicted in FIGS. 5A and 5B and how the reaction vessel can include a channel: for guiding solution hack and forth between the two discrete regions;

[00311 FIGS, 6A-6C show examples of hybridization and solid-phase PCK operations in a reaction vessel having a light absorbing layer with _: multiple discrete regions;

[00321 FIG. 6 shows another example of solid phase PCR in which single strands of DMA are bonded to single strand DMA atached to discrete regions of a light absorbing layer

[00331 F IR 7 A shows a top view of a portion of a reaction vessel having a reaction vessel wall formed from optically transparent material; [00341 FIG. 7B shows a second configuration of a light absorbing layer;

[00351 FIG, 7C shows a third configuration of alight absorbing layer in which a central region of light absorbing layer defines an electrically conducti ve pathway and a peripheral region of the light absorbing layer is used only for heat generation and transfer;

[00361 FIG· 7D shows a fourth configuration of light absorbing layer hi which am electrically conduct i ve pathway formed by ii ght absorbing layer is concentrated in multiple discrete regions;

[00371 FIG, 8 shows a reaction vessel with a light absorbing layer distributed across a reaction vessel wall;

[00381 FIG. 9.A shows a top v iew' of a portion of a reaction vessel having a light absorbing layer arranged upon a reaction vessel wall;

[0039Ϊ FIG. 9B shows a cross-sectional view of a reaction vessel in accordance with section line A-A of FIG. 9A

[00401 F IG. 10 shows a feedback control loop for modulating the output of a photoni c energy source into a reaction vessel based on temperature sensor readings; [00411 FIGS. HA -1 1C show different light absorbing layer configurations formed from a patterned metallic fil that include discrete regions arranged so that a density of the light absorbing layer varies across a surface of a reaction vessel wall; and:

[00421 FIGS- 1 ID~11 E show exemplary side views of a distribution of heat within reaction vessels using conventional light absorbing layers and patterned light absorbing layers. [00431 FIG. 12 shows an example method for operating a reaction vessel assembly.

DETAILED DESCRI PTION OF THE INVENTION

[00441 In the following detailed description, references are made to the accompanying drawings, which form a part of the description and in which are shown, by way of illustration, specific embodiments in accordance with the described embodiments. Although these embodiments are described in sufficient detai l to enable one skilled in the art to practice the described embodiments, it is understood that: these examples are not limiting; such that other embodiments may be used, and changes may be made without departing from the spirit and scope of th e described embodiments,

[0045j icroftiiidics systems or devices have widespread use in chemistry and biology _< In such devices, fluids are transported, mixed, separated or otherwise processed. In many microfltiidics devices, various applications rely on passive fluid control using capillary forces. In other applications, external actuation means (e.g., rotary drives) are used for the directed transport of fluids.“Active microlfuKhes" refers to tire defined manipulation of the working fluid by active (micro) components such as mieropumps or micro valves.

Micropumps supply fluids in a continuous manner or are used for dosing. Microvalves determine the flow direction or the mode of movement of pumped liquids. Processes that are normally carried out in a laboratory can be miniaturized on a single chip in order to enhance efficiency and mobility as well to reduce sample and reagent volumes. Microflnidic structures can include micropneumatic systems, Le., microsystems for the handling of off- chip fluids (liqui pumps, gas valves, etc.), and microfluidic structures for the on-chip handling of nanoliter (nl) and picoliter (pi) volumes (Nguyen and Wereley, Fundamentals and Applications of Microfluidics, Artecli House, 2006). [0046J Advances in mierofluidi es technology are revolutionizing molecular biology procedures for enzymatic analysis (e.g., glucose and lactate assays), DN A analysis (e.g., polymerase chain reaction and high-throughput sequencing), and proteomics. Microfluidic hiochips integrate assay operations such as detection, as well as sample pre-treatment and sample preparation on one chip (Meroid and R&sooly, editors, Lab-on~a~Chip Technology· Fabrication and Mierofiuidics, Caister Academic Press, 2009· Herold and Rasooly, editors,

Lab-on-a-Chip Technology: Btomolecular Separation and Analysis, Caister Academic. Press, 2009). An emerging _: application area for bioehips is clinical pathology, especially the immediate pomt-of-eare di agnosis of diseases. In addi tion, some mierotluidics-based devices are capable of continuous sampling and real -time testing of atr/water samples for biochemical toxins and other dangerous pathogens.

[00471 Many types of microfluidic architectures are currently in use and include open microfluidics, continuous- flow microfluidics, droplet-based microfluidics, digital

microfluidics, paper-based microfluidics and DMA chips (microarrays).

[00481 Id open microflui dics, at least one boundary of the system is removed, exposing the fluid to air or another interface (he.. liquid) (Berthier et ah, Open microfluidics, Hoboken,

NJ: Wiley, Scrivener Publishing, 2016; Pfoh! et ah, Ghent Phys Chet»; 4:1291-1298, 2003; aigala et ah, Angewandte Ghemie Internationa] Edition. 51:11224— 11240, 2012).

Advantages of open mierofluidics include accessibility to the flowing liquid for intervention, larger liquid-gas surface area, and minimfoed bubble formation (Berthier et ah, Open microfluidics, Hoboken, NJ; Wiley, Scrivener Publishing, 2016; Kaigala et ah, Ange. Ghemie Int, Ed. 51:1 1224-1 1240, 012; Li efc al., Lab on a Chip 17: 1436-1441). Another advantage of open microfluidics is the ability to integrate open systems with surface-tension driven fluid flow, which eliminates the need for external pumping methods such as peristaltic or syringe pumps (Casavant et al., Proc, Nat. Acad. Sci. PSA 110:101 11-10116, 2013). Open microfluidic devices are also inexpensive to fabricate by milling, ther ofonriirtg, and hot embossing (Guekenberger et al., Lab on a Chip, 15 : 2364-2378, 2015; Tmckenmuller et ah, j. Miero eehanics and Microengineering, 12: 375-379, 2002; Jeon et ah, Biomed,

Microdevices 13; 325-333, 2010; Young et ah, Anal. Chem S3 '1408-1417, 2011). jn addition, open microfluidics eliminates the need to glue or bond a cover for devices which could be detrimental for capillary flow's. Examples of open microfteidics «delude Open- channel micro fluidics, rail-based microfluidics, paper-based, arid thread-based: mierofliudics (Berthier et ah, Open microfiuidies, Hoboken, NJ: Wiley, Scrivener Publishing, 2016;

Casavant et ah, Proc, Nat. Acad. Sci. USA 110:10111-10116, 2013; Bouaid t et ah, Lab on a Chip 5: 827, 2005).

[0049] Continuous flow microfluidics are based on the manipulation of continuous liquid flow·' through inierolbbricated channels (Nguyen et ah, Micromachines 8: 1:86, 2017; Antfolk and Laurdl, Anal. Chim. Aeta 965:9-35. 2017), Actuation of liquid flow is implemented either by external pressure sources, external mechanical pumps, integrated mechanical micfopumps, or by combinations Of capillary forces and eleetrokinetie mechanisms, Continuous-flow devices are useful for many well-defined and simple biochemical applications and for certain tasks such as chemical separations, but they are less suitable for tasks requiring a high degree of flexibili ty or fluid manipulations, Process monitoring capabilities in continuous-flow systems can be achieved with highly sensitive microfiuidic flow sensors base on micro-eiectro-mechameal systems (MEMS) technology, which offers resolutions down to the nanoliter range.

(O05OJ Droplet-based mierofluidics manipulates discrete volumes of fluids in immiscible phases with low Reynolds number and laminar flow regime (see reviews at Shembekar et a!„ Lab on a Chip 8:1314-1331, 2016; Zhao-Miao et ah, Chinese 3. Anal. Chem. 45:282-296, 2017. Microdroplets allow for the manipulation of miniature volumes (pi to fl) of fluids conveniently, provide good mixing, encapsulation, sorting, and sensing, and are suitable for high throughput applications (Chokkalingam et al, Lab on a Chip 13:4740-4744, 2013).

[00511 Alternatives to closed-channel continuous-flow systems include open structures, wherein discrete, independently controllable droplets are manipulated on a substrate using electrowetting, By using discrete unit-volume droplets (Chokkalingam et al., Appl. Physics Lett. 93:254101, 2008), a microfluidie function can be reduced to a set of repeated basic operations, i.e., moving one unit of fluid over one unit of distance. Hits ^digitization” metbod facilitates the use of a Hierarchical , cell-based approach for microfiuidic; biochip design. Therefore, digital nncrofiuidies offers a flexible, scalable syste architecture as well as high iau t-tolerartce. Moreover, because each droplet Can be controlled independently, these systems also have dynamic reconfigurability, whereby groups of unit cells in a microfiuidic array can be reconfl gured to change their functionality dinin the concurrent execution of a set of bioassays. Alternatively, droplets can be manipulated in confined microfluidie channels _* One common actuation method for digital mierofluidics is electro wetting-on- dielectric (EWQD) (reviewed in Kelson and Kim, 3. Adhesion Sei. Tech,, 26; 12- 17, 1747- 1771 , 2012). Many lab-on-a-ehip applications have been demonstrated withi the digital microfluidics paradigm using eleetrowetiing. However, recently other techniques for droplet manipulation have also been demonstrated using magnetic force (Zhang and Nguyen, Lab on a Chip 17.6: 994*1008, 2017), surface acoustic waves, optoeleetfowetting, mechanical actuation (Sbemesh et ah, Biorned. Mierodeviees 12:907-914, 2010), etc.

[00521 Paper-based mierofluidics (Berthier et al., Open Mierofluidics, John Wiley & Sons, Inc. pp„ 229-256, 2016) rely on the phenomenon of capillary penetration in porous media, Ill order to tune fluid penetration in porous substrates such as paper in two and three dimensions, the pore structure, wettability and geometry of the microtluidic devices can be control led, while the viscosity and evaporation rate of the liquid play a further signi ficant role. Many such devices feature hydrophobic barriers oh hydrophilic paper that passively transport 5 aqueous solutions to outlets where biological reactions take place (Ga!lndo-Rosales, Complex Fluid-Flows in Microti uidics, Springer. 2017).

((10531 Early Mochips were based on the idea of a DNA microarray, e.g,, the GeneChi DNA array from Affymetrix, which is a piece of glass, plastic or silicon substrate on which DNA molecules (probes) are affixed in an array. Similar to a DNA niicroarray, a protein:0 array is an array: in which a multitude of different capture agents, e.g., monoclonal antibodies, are deposited on a chip surface. The capture agents are used to determine the presence and/or amount of proteins in a biological sample, e.g,, blood. For a review, see, e.g., Bumgarner, Curr. Ptotoo. Mo!. Biol. 101 :22.1.1 -22.1 ,11 , 2013.

10054} In addition to raicroarrays, bioehips have been designed for two-dimensional 5 electrophoresis, tramcriptome analysis, and PCR amplification. Other applications include various electrophoresis and liquid chromatography applications for proteins and DNA, cell separation, in particular, bloo cell separation, protein analysis, cell manipulation and analysis including cell viability analysis and microorganism capturing.

[00551 Reaction vessels are often use to perform various types of operations on DNA0 strands that include polymerase chain reactions (PCR) and DNA sequencing. Reaction

vessels can incorporate one or more of the microfluidie architectures listed above but it should be appreciated feat reaction: vessels can be larger than mierofluidies devi ces and for that reason may not incorporate any of fee microfluidic architectures describes above.

Operations of the reaction vessels often include the need to make rapid changes in

5 temperature withi the reaction vessel. For example, a PCR operation solution containing DNA strands is positioned within a reactio chamber defined by the reaction vessel. A heating element is use to thermally cycle the solution in order to breakdown and/or build up different types of DMA. Unfortunately, conventional means of thermally cycling the solution are often slower than desire and not capable of varying a temperature of specific regions of a0 reaction chamber wi thin the reaction vessel.

(0056) One solution to this problem is to position a light absorbing layer within the reaction chamber of fee reaction vessel with light absorption characteristics that allow absorption of between 50 and 90% of the photonic energy in any light absorbed by the light absorbing layer. An energy source can be configured to direct light at the light absorbing layer, which efficiently absorbs energy from photons of the light directed at the light absorbing layer. The absorption of the photonic energy rapidly increases the temperature of the li ght absorbing 5 layer. This energy received by the light absorbing layer is then transferred to a solution within the reaction chamber by thermal conduction.

[00571 hi some embodiments, the light absorbing layer is divided into discrete regions. Dividing the light absorbing layer into discrete regions has the following advantages: ( 1) patterning tire discrete regions into different shapes and thicknesses allows a specific spatial:0 heating profile to be achieved within the reaction chamber of the reaction vessel ; (2) optical sensors are able to take readings of solution wi thin the reactio chamber through gaps between the discrete regions; and (3) an array of energy sources can be used to add different amounts of energy to each of the discrete regions of the light absorbing layer, thereby allowing solution within a first region of the reactio chamber to have a ubstantially5 different temperature than solution withi a second region of the reaction chamber.

[00581 hi some embodiments, the light absorbing layer can be patterned as a serpentine or meandering electrically conductive pathway that covers a majority of a light absorbing surface of the reaction vessel. A temperature of the reaction vessel can be continuously monitored by routing electrical current through this electrically conductive pathway, A0 resistance of this electrical! y conductive pathway to electricity ca be correlated with a

temperature of the reaction chamber. In this way, the light absorbing layer is operative to convert photonic energy into heat energy within the: reaction vessel an monitor a temperature of the reaction vessel. In some embodim ents, the temperature data derived from the measured electrical resistance can be used to perform feedback control of the amount of 5 photonic energy directed at the 1 ight absorbing layer to achieve a desired thermal profile within the reaction chamber. In some embodiments, the reactio chamber can include a first light absorbing layer patterned as an electrically conductive pathwa and a second light absorbing layer that operates only to eat material within the reaction: vessel in some embodiments, the first and second layers ean have substantially conformal shapes that0 prevent the presence of large gaps between the layers that could lead to uneven heating of the reaction chamber.

I I [0O59J These and other embodiment are discussed below with reference to FIGS. 1 A- 10; however, those skilled in the art will readily appreciate that the detailed description: give herein with respect to these figures is for explanatory purposes only and should not be construed as limiting. More information about various aspects of light absorbing layers, reaction vessels, and the moni toring of the reaction vessels is disclosed in U.S. Patent

Application No, 16/653,734, filed October 15, 2019; and U.S. Patent Application No.

16/654,462, filed October 16, 2019; the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

[OOfiOj FIG. I A shows a perspective view of an exemplary reaction vessel 100 suitable for use with the described embodiments. In particular, the reaction vessel 100 includes a housing component 102 forme from an Optically transparent material that defines a reaction chamber 104. While reaction chamber 104 is depicted as having a substantially eitculargeomefry it should be appreciate that the depicted shape of reaction chamber 104 should not be construed as limiting and other shapes such as oval, rhombic and rectangular are also possible. In some embodiments, the optically transparent material forming housing component 102 can be optically transparent to only those wavelengths of light that are use to heat reaction vessel 100. For example, the optically transparent material could be optically transparent to only select visible, infrared or ultraviolet frequencies of light Reaction chamber 104 can be closed by a second housing component (mot depicted) that encloses a liquid being heated within reaction chamber 104. In this way, DMA strands in a liqui solution within reaction chamber 104 can undergo rapid thermal cycles ant! at least a portion of any vaporized portion of the solution can subsequently condense back into the solution between the thermal cycles or after the thermal cycling is complete, A light absorbing layer 106 can be plated onto or otherwise adhered to an Interior-faemg surface of the reaction chamber 104. Light absorbing layer 06 has good light absorbing properties an can be in direct contact with any liquid disposed within reaction chamber 104. For example, light absorbing layer 106 can be configured to absorb about 50-90% of the p hotonic energy incident to light absorbing layer 106. In some embodiments, light absorbing layer 106 can be a metal film formed from elemental gold, chromium, titanium, germanium or a gold alloy such as, e.g , gold-germanium, gold-chromium, gold-titanium, gold-chromium-gennanium, and go id-t I tain um-ge nn an i u in . in some embodiments, light absorbing layer 106 cam he a multilayer metal film formed from elemental gold, chromium, titanium, germanium or a goldalloy such as, e.g., gold-germanium, gold-chromium, gold-titanium, giild-chromium- germanium, and gold-titanium-germanium. light absorbing layer 106 can have a thickness of about 5n -20djftnL Housing component 102 also defines inlet channel 108 and outlet channel 1 10, which can be used to cycle various chemicals, primers, DMA strands, and other biological materials into and Out of reaction chamber 104, In some embodiments, housing component 152 can have dimensions of about 7mm by 14min; however, it should be

appreciated that this size can vary.

(00611 FIG. IB shows a perspective view of another exemplary reaction vessel 150. The reaction vessel 150, similar to the reaction vessel 100 includes housing component 152, reaction chamber 104, light absorbing layer 106, the inlet channel 108 and outlet channel 110. Housing component 152 includes a widened central region that accommodates the inclusion of air gap regions 154 and 156, Air gap regions 154 and 156 can he left em ty in order to discourage the lateral transmi ssion of heat to adjacent reaction vessels. In some embodiments, the transfer of heat through air gap regions 154 and 156 ca be further reduced by removing the air from air gap regions 154 and 156. I some embodiments, a diameter of housing component 152 can be about 5mm; however, it should be appreciated that this si^e can vary. For example, the diameter of housing component 152 could vary from 2mm to 15 mm.

(0062J FIG. 1C shows how (he shape of housing component 152 allows reaction vessels Ϊ 50 to be packed tightly into a honeycomb or hexagonal pattern. FIG:. 1C also illustrates how air gap regions 154 and 156 are able to establish robust: barriers that reduce the lateral transfer of heat between adjacent reaction vessels 150. When a diameter of the reaction vessel 150 is about 5mm reaction chamber 104 can behold about lOul of solution and have a depth of BOOum. Generally, these devices are configured to hol between 2.5ul and 500ul with a depth of 200-1500um,

[0063j PIG. 2 shows a schematic cross-sectional side view of a reaction vessel 100 and how reaction chamber 1 i)4 defined by housing component 102 can he closed b housing component 202, which can take the form of a cap. In some embodiments, housing

components 102 and 202 can be sealed together to prevent contamination and allow for control of other factors such as pressure within reaction chamber 104. FIG. 2 also shows energy source 204, which is configured to project light upon light absorbing layer 106. A frequency of the light projected by energy source 204 can vary. In some embodiments, energy source 204 can take the form of a light: emitting diode configured to emit light ith a wavelength of 45 Ohm, a power of 890rnW and current of a 700mA. When light absorbing layer 106 is illuminated by an energ source, a large temperature difference between the hot metal surface and the cooler surrounding solution disposed withi reaction chambe 104 occurs, resulting in the heating of the surrounding solution. When the energy source stops 5 illuminating light absorbing layer 106, the resultin rapid cooling of the light absorbin layer 106 helps facilitate rapid cooling of the heated solution.

10064! FIG, 3A shows a cross-sectional side view of reaction vessel 100 and how light absorbing laye 106 can be separated into discrete regions 302, 304 and 306. In some embodiments, these discrete regions can be set up to help establish a targeted amount of:0 energy into reaction chamber 104. The gaps between discrete regions 302, 304 and 306 reduce a total surface area across which light is received from energy source 204 compared with a light absorbing layer that extends across an entire bottom surface of the reaction chamber 104, Increasing or decreasing the size of the gaps between discrete regions 302, 30 an 306 can he used to tune the energy input into reaction chamber 104. A total area in5 contact with solution within reaction chamber 10 is also reduced, thereby reducing an

efficiency of the transfer of heat fro discrete regions 302, 304 and 306 to th solution. Gaps between discrete regions 302, 304 and 306 also allow for optical monitoring of soluti on within reaction chamber 104 Gaps between discrete regions 302, 304 and 306 may not be uniform in size allowing for some discrete regions within reaction chamber 104 to be heate0 substantially more than other discrete regions. Furthermore, discrete region 306 can be

thicker than discrete regions 302 and 304, thereby increasing the efficiency with which heat can be drawn into reactio chamber 104 proximate discrete region 306.

[0065| FIG _* 3B shows a cross-sectional side view of reaction vessel 100 being illuminate by an array of energy sourcesJOB Using an array of energy sources 308 can reduce an5 amount of light extending between discrete regions 302-306 by allowing energy sources 308 to be focus energy only on discrete regions 302-306. In some embodiments, energy sources 308 may include specialized focusing optics to specifically target one of the discrete regions 302, 304 or 306 Each energy source 308 of the array of energy sources 308 can be controlled separately to create a desire gradient of heat within reaction chamber 104. For example,0 different types: of biological material can be attached proximate: or directly on top of a

particular one of the discrete regions 302-306. Because energy sources 308 can he controlled individually, the materials associated with a particular discrete region can be heated in accordance with a customized heating profile. For example, biological material proximate discrete region 306 could have a substantially lower denaturi ng temperature than the biological material proximate discrete region 302, By operating energy source 308-1 at a higher power level than the energy source 308-3 a desired denaturing temperature can be achieve for both types of biological material.

5 [00661 FIG. 3C shows a eross-seetional side view -of reaction vessel 100 being illuminated by an arra of energy sources 308, FIG . 3C shows how housing component 202 can include multiple protrusions or ridges 303 that meet protrusions or ridges 305 of housing component ! 02 to divide reaction chamber 104 into multipie smaller reaction chambers 104- 1, 104-2: and .1.04-3. in this way, the solution within reaction chamber 104 can be separated* further:0 improving the thermal isolation enabled by discrete regions 302, 304 and 306. While both bousing components 202 and 102 are shown including respective protrusions 303 an 305, it. should be appreciated that in some embodiments, protrusions 303 could extend all the way to a flat interior-facing surface of housing component 102 or protrusions 305 could extend all the way to a fiat interior-facing surface of housing component .202, In some embodiments, the5 reaction vessel 100 could include multiple different housing components 202 with different configurations of protrusions 303. For example, a housing component 202 with no protrusions could allow reactions to be carried out with a single reaction chamber 104 and in subsequent experiments or operations, the depicted housing component 202 with protrusions 303 could divide the reaction chamber into multiple smaller reaction chambers as depicted. In0 other embodiments, housing component 202 could include a configuration of protrusions 303 that defined different sized reaction chambers 104. For example, housing component 202 could include only one protrusion 303 defining one reaction chamber 104 that includes discrete regions 302 an 304 and then another reaction chamber 104 that includes only discrete region 306. It should be appreciate that protrusions 303 and/or 305 can include5 sealing: elements at their distal ends that help prevent the passage of solution between

adjacent reaction chambers 104.

[00671 MG, 3D shows a cross-sectional side view of a thermal profile of a portion of a reaction vessel with different sized gaps between, iscrete regions 302, 304 and 306 of a light absorbing layer. In particular, housing component 102 is depicted with four different discrete0 region configurations* which are differentiated by: the labels housing component 102-1 , 102- 2, 102-3 and 102-4. These configurations depict two sets of contours indicative of an amount of energy or temperature change taking place in portions of the solution adjacent to discrete regions 302-306, In particular, these depictions show how· adjusting a gap size between adj acent di screte regions can i mpro v e or change a uniformity of the heating appli ed to a solution within a reaction chamber. Housing component 102-4 shows Only a large single discrete region or alternatively, a discrete region made u of regions 302, 304 and 306 in abutting contact with one another such that they effectively form a single discrete region. It should be noted that while placing discrete regions 302-306 in abutting contact yields the largest heated area in a central portion of housing component 102-4, the peripheral ends of housing component 102 can fall below a desired temperature in some embodiments,

[00681 FIG. 4A shows a schematic perspective view ofa; reaction vessel 400 that includes housing component 402 Housing component 402 includes a light absorbing layer 404 distributed into multiple discrete regions 406 configured to receive optical radiation from energy source 408 for the localize heating of solution disposed within reaction vessel 400, Housi ng component 402 has a reaction chamber taking the for of a serpenti ne channel through which solution can flow through each of the discrete regions 406, The flow of solution through serpentine channel 410 can be facilitated in many ways including by a pump, by a winking structure or the flow may be gravity-fed, It shoul be appreciated that each of the discrete regions 406 can also be configured with its own respective energy source 408 similar to the configuration depicted in FIG 3B.

[00691 FTG. 4B shows a to view of the reaction vessel 400 with labels indicating each of discrete regions 406-1 through 406-8 of light absorbing layer 404, While FIG. 4B shows a direction of the flow of solution through serpentine channel 410 in a first direction, it should be appreciated that the flow of solution through serpentine channel 410 can move in a second direction opposite the first direction. In: embodiments that include a pump mechanism, tire flow of solution through serpentine channel 410 can be reversed at various points during a reaction to achieve a desired thermal heating profile for the solution disposed within channel 410. In some embodiments, single- stran DMA e an be affixe to binder positioned atop one or more of discrete region s 406-1 allowing biol ogical materials within the soluti n being conducted along channel 410 to interact with tire single-strand DNA at various temperatures generated by heat transferred to the solution at discrete regions 4:06. in some embodiments, a speed at Which the solution passes through channel 410 can be varied by increasing the width and or depth of the ehairneL For example, channel segment 412 is depicted having an increased width thereby reducing the speed and increasing the time the solution has to cool between discrete regions 406-1 and 406-2. [0070J FIG. 4C shows another embodiment of reaction vessel 400 that includes a light absorbing layer with conformal discrete regions 414 Conformal discrete regions allow the energy from an energy source to be targeted at specific sections of channel 410 In this way, a length of segments of channel 410 between sequential discrete regi ons can be increased or decreased. For example, as depicted solution flowing between regions 414-1 , 414-2 and 414- 3: has less time to cool than when the solution is passing from region 414-3 to region 414-4 As previous! y describ ed, each of discrete regions 414 could be supported by a shared energy source, by its own dedicated energy source, or by an energy source that illuminates a subset of discrete regions 414; It should be appreciated that while reaction chambers have been described in the context of a unitary chamber as shown in FIGS. 1 A-3B, a di vided reaction chamber as shown in FIG. 3C and as a channel in FIGS. 4A-4G that other reaction chamber configurations are possible. For example, a reaction chamber coul d take the form of an interior volume defined by a series of glue channels positioned between two flat plates or could simply consist of a location on a reaction vessel su bstrate. In general, the reaction chamber can be considered to be any fluidic path defined by the reaction vessel along which various reactions can be initiated. The fluidic path could be closed/sealed or open to the environment in certain embodiments.

[00711 FIG. 5A shows a reaction vessel 500 that includes a housing component 502 having a light abs orbing layer made up of two discrete regions 504- 1 and 504-2. In some embodiments, discrete regions 504-1 and 504-2 can be driven by a single energy source 506, allowing each of discrete regions 504 to receive similar amounts qf energy, FIG, SB shows how energy source 506 can be offset toward discrete region 504- 1 such that discrete region504-1 receives more energy from energy source 506 than discrete region 504-2 This variance in energy between discrete regions 504-1 and 504-2 can be increased more by _: reflector element 508 that further limits the amount of light arriving: at discrete region 504-2 and is able to increase the light arriving: at discrete region 504- 1 by reflecting light emitted by- energy source 506 toward discrete region 504-1 as depicted. In some embodiments, reflector element 508 can be tilted toward discrete region 504- i to further increase the amount of light received from energy source 506.

[00721 FIG. SC shows a top view of reaction vessel 50Q and how it can include a channel 5 _: 10 for guiding solution back: and forth _: between discrete regions 504-1 and 504-2 In some embodiments, discrete regi ons 504-1 and 504-2 can: recei ve the same amount of energy. In this ty e of heating configuration, solution flowing through channel 510 passes from discrete region 504-1 at time Ti, through a bortion of channel 510 disposed between discrete regions 504-1 and 504-2 at time ¾ and then: through discrete region 504-2 at time ¾. In this way the solution carried by channel 510 cycles from a first temperature at time Ti to a second temperature at time T2 and then back to the first temperature at time ¾. In other

embodiments, discrete regions 504-1 an 504-2 can receive different amounts of energy by offsetting an associated energy source toward one of discrete regions: 504 by including dedicated energy sources for each of discrete regions 504 or by increasing or decreasing a thickness of a portion of the light absorbing layer making up one of discrete regions 504. By configuring the system to provide different amounts of energy at discrete regions 504-1 and 504-2, solution flowing through channel 510 is able to reach a larger variety of temperatures as it flows from one end of ch annel 510 to another.

Exemplary PCR Reactions

[00731 OCR amplifies a specific region of a DMA strand (the DM target). Most PCR methods amplify DNA fragments of between 0.1 and 10 kilo basepairs (kb). The amount of amplifie product is determined by the available substrates in the reaction, which become limiting as the reaction progresses. A basic PCR set-up requires several components and reagents, including: a DMA template that contains the DMA target region to am lify; a DMA polymerase, an enzyme that polymerize new DNA strands; beat-resistant Taq polymerase is especiall common, as it is more likely to remain intact during the high-temperature DMA denaturation process' two DNA primers that: are: complementary to the 3' ends of each of the sense and anti-sense strands of the DNA target; specific primers that are complementary to the DMA target region are selected beforehand, and are often custom-made in a laboratory or purchased from cortupemiaj biochemical suppliers; deoxynueieoside triphosphates, or dHTPs; a buffer solution providing a suitable chemical environment for optimum activity and stabili ty o f the DN A polymerase; hi valent cations, typically magnesium (Mg) or manganese

(Mn) ions; Mg2÷ is the most common, but Mn2+ can be used for PCR-mediated DM A mutagenesis, as a higher Mn2+ concentration increases the error rate during DNA synthesis; and monovalent cations, typically potassium (K) ions.

{00743 The reaction is commonly carried Out in a volume of 10-200 m! in small reaction chambers (0, 2-0,5 ml volumes) in a thermal cycler, which heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction, Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. [0075f Typically, PCR consists of a series of 20-40 repeate temperature changes, called cycles, with each cycle commonly consisting of two or three discrete temperature steps. The cycling is often preceded by a single temperature step at a very high temperature (>90 °€

[194 ^S F]), .followed by one hold at the end for final product extension or brief storage. Th temperatures used and the length of time they are applied in each cycle depend on a variety of parameters, including the enzyme used for DNA synthesis, the concentration of bivalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers. The individual steps common to most PCR methods are as follows:

[0076] (1) Initialization: This step is only required for DMA polymerases that require heat acti vation by hot-start PCR. It consists of heating the reaction chamber to a temperature of 94-96 °C (201-205 ^S F), or 98 °C (208 °F) if extremely iher ostttble polymerases are used, which is then held for 1-10 minutes,

10077J (2) Denaturation: This step is the first regular cycling event an consists of heating the reaction chamber to 94-98 °C (201—208 °F) for 20-30 seconds. This causes DNA melting, or denaturation, of the double-stranded DNA template by breaking the hydrogen bonds between complementary' bases yielding two single-stranded DNA molecules.

[0078J (3) Annealing: In the next step, the reaction temperature is lowered to 50-65 °C

(122-149 °F) for 20-40 seconds, allowing annealing of the primers to each of the single- stranded DNA templates. Two different primers are typically included in the reaction mixture: one for each of the two single-stranded complements containing the target region. The primers are single-stranded sequences themselves, but are much shorter than the length of the target region, complementing only very short sequences at the 3' end of each strand.

The correct temperature for the annealing step is important, since this temperature strongly affects efficiency and specificity. This temperature must be low enough to allow tor hybridization of the primer to the strand, but high enough for the hybridization to be specific, Le., the primer should bind onl to a perfectly complementary part of the strand, and nowhere else. If the temperature is too low, the primer may bind imperfectly. If it is too high, the printer may not bind at all. A typical annealing temperature is about 3— 5 °C below the T of the primers used. Stable hydrogen bonds between complemeniary bases are forme only when the primer sequence very closely matches the template sequence. During this step, the polymerase hinds to die primer-template hybrid and begins D A formation. [007 J (4) Extemien/eloiiigaOoiv: The temperature at this step depends oft the DNA polymerase used; the optimum activity temperature for the thermostable DNA polymerase of Taq (Thermos aquaticos)polymerase is approximately 75-80 °€ (167-176 °:F), though a temperature Of 72 °.C (162 °F) is commonly used with this enzyme. in this step, the DNA 5 polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs fro the reaction mixture that are complementary to the template in the 5 -to-3' direction, condensing the S-phosphate grou of the dNTPs with the 3 '-hydroxy group at the end of the nascent (elongating) DNA strand. The precise time required for elongation depends bot on the DNA polymerase used and on the length of the DNA t arget region to :tO amplify As a rule of thumb, a their optimal temperature, most DN A polymerases polymerize a thousand bases per minute. Under optimal conditions .e., if there are no limitations due to limiting substrates or reagents), at each e tenskm/elongation step, the number of DNA target sequences is doubled. With each successive cycle the original template strands plus ail newly generated strands become template strands for the next round of elongation, leading to 15 exponential (geometric) amplification of the specific DNA target region.

100801 The processes of denaturatkm, annealing and elongation constitute a single cycle. Multiple cycles are required to amplify the DNA target to millions of copies. The formula used to calculate the number of DNA copies formed after a given number of cycles is 2n, where n is the number of cycles.

20 [00811 (5) Final elongation: This single step is optional, but is performe at a temperature of 70-74 °C (158-165 °F) (the temperature range required for optimal activity of most polymerases used in PCR) for 5 -15 minutes after the last PGR. cycle to ensure that any remaining single-stranded DNA is fully elongated,

[0082} (6) Final hold: The final step cools the reaction chamber to 4—15 °C (39-59 °F) for 5 an indefinite time, and may be employed for short-term storage of the PCR products.

[0083| To check whether the FOR successfully generated the anticipate DNA target region (also sometimes referred to as the ampltmer or amplicon), agarose gel electrophoresis may be employed for size separation of the PCR products, The size(s) of PCR products is determined by comparison with a DNA ladder, a molecular weight marker which contains 30 DNA fragments of known size ran on the gel alongside the PCR products, As with other chemical reactions, the reaction rat and efficiency of PCR are affected by limiting factors. Thus, the entire PC process can farther be divided into three stages based on reaction progress:

[00841 ( 1.) Exponential amplification; At every cycle, the amount of product is doubled

(assuming 100% reaction efficiency). After 30 cycles _» a single copy of ON A can be increased 5 tip to one bil lion copies. The reaction is very sensitive: only minute quantities of DMA: must be: present.

100851 (2) Leveling off stage; The reaction slows as the DNA polymerase loses activit and as consumption of reagents Such as dNTFs and primers causes them to become limiting.

[00861 (3) Plateau: No more product accumulates due to exhaustion of reagents and0 enzyme.

[00871 Upon loading and sealing, the system may generate an amplified product through thermal cycling. Thermal cycling may comprise one or more cycles of incubating a reactio mixture at a denafuration temperature for a denaturation time period followed by ineubating the mixture at an annealing temperature for an annealing time period further followed by 5 ineubating tire mixture at an elongation temperature for an elongation time period. A system may heat the wells of the reaction well by using one or more light sources as previously described. Focused light by lens between light source and reaction well may be use also.

The embedde lens may be used to focus emission from the fluorescent dye integrated in the reaction vessel/wells. For the cooling of the sample and reagents, the one or more light(5 sources may be turned off for a cooling time period. In some eases, a fluid circulation channel may be used as previously described for th cooling of the reagents an samples in the Wells of the reaction well

[00881 Amplification of a sample ma be perforated by using the systems described previously to perform one or more thermal cycles comprising a denaturation cycle, an5 annealin cycle and an elongation cycle. The time in which an amplification reaction may yield a detectable result in the form of an amplified product may vary depending on the target nucleic acid, the sample, the reagents used and the protocol for PCR. In some cases, an amplification process may be performed in less than 1 minute. In some cases, an

amplification process may be performed in about 1 minute to about 40 minutes, In some0 cases, an amplification process may be performed in at least abont fmimite. In some cases, an amplification process may be performed in at most about 40 minutes. In some eases, an amplification process may be performed in about 1 minute to about 5 mirmt.es, about 1 minute to about 10 minutes, about I minute to about 15 minutes, about 1 minute to about 20 minutes, about 1 minute to about 25 minutes, about 1 minute to about 30 minutes, about 1 minute to about 35 minutes, abou 1 minute to about 40 minutes, about 5 minutes to about tO minutes, about 5 minutes to about 15 minutes, about 5 minutes to about 20 minute , about 5 brutes to about 25 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 35 minutes, about 5 inutes to about 40 minutes, about 10 minutes to about 15 minutes . about 10 minutes to about 20 minutes, about 10 minutes to about 25 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 35 minutes, about 10 minutes to about 40 minutes, about 1:5 minutes to about 20 minutes, about i 5 minutes to about 25 minutes, about 15 minutes to about 30 minutes, about 15 minutes to about 35 minutes, about 15 minutes to about 40 minutes, about 20 minutes to about 25 minutes, about 20 minutes to about 30 minutes, about 20 minutes to about 35 minutes, about 20 minutes to about 40 minutes, about 25 minutes to about 30 minutes, about 25 minutes to about 35 minutes, about 25 minutes to about 40 minutes, about 30 minutes to about 35 minutes, about 30 minutes to about 40 minutes, or about 35 minutes to about 40 minutes. In some eases, an amplification process ma be performed in about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, or about 40 minutes.

{0089} In some cases, ampli fication of a sample may be performed by repealing the thermal cycle 5 to 40 times. In some eases, the; thermal cycle may be repeated at least 5 times. In some cases, the thermal _: cycle may be repeate at most 60 times. In some eases, the thermal cycle may be repeated 5 times, 10 times, 15 times, 20 times, 25 times, 30 _: times, 35: times 40 times, 45 times, 50 times, 55 times or 60 times.

[0090! A thermal cycle may be completed in a thermal cycling time period , In some eases, a therma cycling: time period may range from 2 seconds to 60 seconds per cycle. In some cases, a thermal cycle may be completed in about 2 seconds to about 60 seconds. In some cases, a thermal cycle nlay be completed in at least about 2 seconds in some cases, a thermal cycle may be complete in at most about 60 seconds in some cases, a thermal cycle may be completed in about 2 seconds to about 5 seconds, about seconds to about 10 seconds, about 2 seconds to about 20 seconds, about 2 seconds to about 40 seconds, about 2 seconds to about 60 seconds, about 5 seconds to about 10 seconds, about 5 seconds to about 20 seconds, about

5 seconds to about 40 seconds, about 5 seconds to about 61) seconds, about 10 seconds to about 20 seconds, about 10 seconds to about 40 seconds, about 10 seconds to about 60 seconds, about 20 seconds to about 40 seconds, about 20 seconds to about 60 seconds, or about 40 seconds to about 60 seconds. In some cases, a thermal cycle may be completed in about 2 seconds, about 5 seconds, about 10 seconds, about 20 seconds, about 40 seconds, or about 60 seconds,

[00911 The temperature and time period of the denaturation cycle may be dependent on the properties sample to be identified, the reagents and the amplification protocol being used. A denaturation cycle may be performed at temperatures ranging from about 80° C; to about 1 10° C. A denaturation cycle may be performed at a temperature of at least about. 80* C. A denaturation cycle may be performed at a temperature of at most about i 10° C, A

denaturation cycle may be performed at a temperature of about 80° C do about 85° C, about W° C to about 90° C, about 80° C to about 95° C, about 80° C to about 100° C, about 80° C to about 105 ^s C, about 80 ^s C to about 110 ^s C, about 85 ^s € to about 90 C, about 85° C to about 95° C, about 85" C to about 100” C, about: 85° C to about 105° C, about 85° C to about 1 10° C, about 90° C: to about 95° C, about 90° C to about 100° C, about 90° G to about 105° C, about 90° C to about 110° C, about 95° C to about 100° C, about 95° C to about 105° C, about 95° C to about 110° C, about 100° C to about 105° C, about 100° C to about 110° C, or about 105° C to about 1 10° C. A denaturation cycle may be performe at a temperature of about 80° C, about 85 ^s C, about 90 ^Q C, about 95 ^s C, about 100 ^s C, about 105° C, or about 1 10° C.

[0092] In some cases, the time period of a denaturation cycle may be less than about 1 second. In some cases, the time period of a denaturation cycle may be at most about 100 seconds. In some cases, the time period of a denaturation cycle may he about 0 second to 1 second, about 1 second to about 5 seconds, about I second to about 10 seconds, about 1 second to about 20 seconds, about 1 second to about 40 seconds, about I second to about 60 seconds, about i second to about 100 seconds, about 5 seconds to about 10 seconds, about 5 seconds to about 20 seconds, about 5 seconds to about 40 seconds, about 5 seconds to about 60 seconds, about 5 seconds to about 100 seconds, about 10 seconds to about 20 seconds, about 10 seconds to about 40 seconds, about 0 seconds to about 60 seconds, about 10 seconds to about 100 seconds, about 20 seconds to about 4:0 seconds, about 20 seconds to about 60 seconds, about 20 seconds to about 100 seconds, about 40 seconds to about 60 seconds, about 40 seconds to about 100 seconds, or about 60 seconds to about 100 seconds.

In some cases, the time period of a denaturation cycle may be less than about 1 second, about 5 seconds, about 10 seconds, about 20 seconds, about 40 seconds, abou 60 seconds, or about 100 seconds. [Q093| The temperature and time period of the annealing and elongation cycles may be dependent on the properties sample to be identified, the reagents and the amplification protocol being used. An annealing and/or elongation cycle may be performed a a temperature of about 40- C to about 70° C. An annealing and/or elongation cycle may be performed at a temperature of at least about 40° C. An annealing and/or elongation cycle may be performed at a temperature of at most about 70° C. An annealing and/or elongation cycle may be perfomied at a temperature of about 40° C to about 45° C, about 40° C to about 50° C, about 40° C to about 55° C, about 40° C to about 60° C, about 40° C to about 65* C, about 40° C to about 70° C, about 45° C to about 50° C, about 45° C to about 55 ^® C, about 45- C to about 60° C, about 45° C to about 65° C, about 5° C to about 70° C, about 0^ C to about 55° C, about 50° C to about 60 ^® C, about 50° C to about 65 ^p C, about 50° C to about 70° C _» about 55* ;C to about 60* C, about 55° C to about 65° C, about 55* C to about 70* C, about 60* C to about 65° C, about 60° C to about 70° C, or about 65" C to about 70° C. An annealing and/or elongation cycle may be performed at a temperature of about 40° C, about 45° C, about 50* C, about 55° C, about 6fF C, about 65° C, or about 70° C.

P094| In some cases, the time period of an annealing and/or elongation cycle may be less than abou t 1 second in some cases, the time perio of an anneal ing and/or elongati on cycle may be at most about 60 seconds in some cases, the time period of an annealing and/or elongation cycle may be about 0 seconds to 1 seconds, about I second to about 5 seconds, about 1 second to about 10 seconds, about I second to about 20 seconds, about 1 secon to about 40 seconds, about 1 secon to about 60 seconds, about 5 seconds to about 10 seconds, about 5 seconds to about 20 seconds, _: about S seconds to about 40 seconds, about 5 seconds to about 60 seconds, about 10 seconds to about 20 seconds, about 10 seconds to about 40 seconds, about 10 seconds to about 60 seconds, about 20 seconds to about.40 seconds, about 20 seconds to about 60 seconds, or about 40 seconds to about 60 seconds, i some eases the: time period of an annealing and/or elongation cycle may be less than about 1 second, about 5 seconds, about 10 seconds, about 20 seconds, about 40 seconds, or about 60 seconds.

[0095 j In some eases, a cooling cycle may be performed between the denaturation cycle and annealing and/or elongation cycles. In some cases, a cooling cycle may be performed for about 1 second to about 60 seconds. In some cases, a cooling cycle may be performe for at least about 1 second. In some eases, a cooling cycle may be performed for at most about 60 seconds. In some cases, a cooling cycle may be: performed for about 1 second to about 5 seconds, about 1 second to about 10 seconds, about I second to about 20 seconds, about 1 second to about 30 seconds, about 1 second to about 40 seconds, about 1 second to about 50 seconds, about 1 second to about 60 seconds, about 5 seconds to about 10 seconds, about 5 seconds to about 20 seconds, about 5 seconds to about 30 seconds, about 5 seconds to about 40: seconds, about 5 seconds to about 50 seconds, about 5 seconds to about 60 seconds, about 10 seconds to about 20 seconds, about 10 seconds to about 30 seconds, about 10 seconds to about 40 seconds, about 10 seconds to about 50 seconds, about 10 seconds to about 60 seconds, about 20 seconds to about 30 seconds, about 20 seconds to about 40 seconds, about 20 seconds to about SO seconds, about 20 seconds to about 60 seconds, about 30 seconds to about 40 seconds, about 30 seconds to about 50 seconds, about 30 seconds to about 60 seconds, about 4 seconds to about 50 seconds, about 40 seconds to about 60 seconds, or about 50 seconds to about 60 seconds. In some eases, a cooling cycle may be performed for about 1 second, about 5 seconds, about 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, or about 60 seconds.

[60961 Detectio of the amplified product may be performed at van ous stages of theamplification process. In some cases, the detection of a amplified product may be performed at the end of the amplification process, in some cases, the detection of the amplified product may be performed during a thermal cycle. Alternatively, In some cases, detection may be performed at the end of each thermal cycle, in addition to the detection methods describe herein, detectio of an amplified product may he performed using gel electrophoresis, capillary electrophoresis, sequencing, short tandem repeat analysis and other known methods.

{0097! FIGS.6A 6C show examples of hybridization and solid-phase PCR operations in reaction vessel 600 having a light: absorbing layer with multiple discrete regions. FIG, 6A shows a top view of a reach on vessel 600 having a light absorbing layer 602 that includes an array of discrete regions 604 arranged in a grid pattern. Reaction vessel 600 includes inlet port 606 and outlet port 608 that are configured to allow solution to How into an out of reactio chamber 609.

{(1098! In some embodiments, one or more of the discrete regions of a reaction vessel may be bound to one or more nucleotide sequences. For exampl e, referencing the example illustrated in FIG. 68, each discrete region 604 possesses unique probes or primer sequences 610, 61 1, 612, and 613 that are bound, for example, by weak eovalent interactions (e.g., Au- thiol). As illustrated, a solution including the target molecules 610’, 611’ and 613’ may be flowe over the discrete regions 604, These probes or primer sequences may he complementary to the target molecules 610’, 61 ;1’ and 613’. In the illustrated example, the solution also includes target molecules 615, Which are not complementary to any of the bound probes or primer sequences. Target mol ecu les 610’ , 611 \ 6 ! 3 ⁵ and 615 ⁵ can be iluorescent ly labele for detectio purposes.

[0099f Id some embodiments, as the solution is caused to flow across the di screte regions 604, target molecules in the solution that are complementary to sequ nces bound to the discrete regions 604 may hybridize with those sequences, FIG. 6C shows how target molecules only hybridize with their complementary sequence hound on the surface. For example, as illustrated in FIG. 6C, target molecules 610’, 63 G and 613’ hybridize with their respective complementary sequences 10, 61 1 and 613 that are themselves bound to their respective discrete regions 604. Thus, target molecules 610’ , 611’ and 13’ become bound to the discrete regions 604 via their respective complementary sequences. Target molecules that do not have complementary sequences (e.g„ the target molecule 61 S’) may not hybridize, and may therefore be left unbound in solution. These unbound molecules may leave the reaction vessel when the solution is caused to flow out of the reaction vessel (e.g _> , referencing FIG, 6A, via the outlet port 608). Similarly, sequences bound to discrete regions 604 that do not have complementary target molecules in solution do not hybridize. For example, as illustrated in FIG. 6C, the sequences _: 612 remain un-hybridized. As mentioned previously, In some embodiments, the target molecules may be labeled for detection purposes. For example, the target molecules may be fluorescentl labeled. Once a solution has been flowed into and out of the reaction vessel (after sufficient time has been aflewed for hybridization), a detection mechanism may be used to detect the presence of target molecules that have been labeled.

For example, the detection mechanism may include a light source designed to excite fluorescent labels bound to target molecules and a camera device ten detecting tlie presence of the fluorescent labels. In this example, the floreseent l ight source: may be shined on the reaction vessel, and the resulting fluorescence intensity at each discrete region can he: used to detect the presence or absence of a _: gi ven target molecule. For example, referencin FIG, 6C, shining a suitable light source at the discrete regions 604 after the solution has been allowed to leave the reaction vessel (along with unbound target molecules such as the target molecule 615’) may cause the fluorescent labels of all hound target molecules to fluoresce. A camera may detect that areas corresponding to discrete regions 604 associated with the sequences 10, 611 a d 613 fluoresce above a threshold intensity, while the area corresponding to the discrete region 604 associated with the sequence 612 does not fluoresce above the threshol intensify (because it lacks target molecules with fluorescent labels).

(0100) Following hybridization, solid phase PGR can be performed to create a population of discrete amplieons, which can then be detected via a molecule that binds to DNA, and can be used to detect the: presence of said molecule (e.g. through fluorescence and/or

electrochemical signal). Solid-phase PGR uses surface-bound primers on the discrete regions instead of freely-diffusing primers to amplify DNA. This may limit the nucleic acids amplification to two-dimensional surfaces on the discrete regions and therefore allows for easy parallelization and high multiplexing of DN A amplification and detection in a singl reaction vessel system. Alternatively, amplieons can be sequenced to identify the presence of said molecule

(01011 FIG. 6B shows another example where each discrete region 604 possesses a unique primer sequence 61 1 ^* bound by weak covalent interactions (e,g., Au-thiol). As illustrated, a solution including target molecules 611, 61 O’ may be caused to flow over the discrete regions 604 We target molecules 611, 610’ may include an adaptor portion 61 1 that is

complementary to the unique primer sequence 61 1:’ an a second portion (e.g·, a portion of a DNA molecule or an RNA molecule). For example, the adapter portion 611 may be an adapter (configured to bind to the unique primer sequence 61 ! ) that is ligated to the second portion 61 Of which may be a DN A molecule. In this example, the adapter portion 61 1 may be foreign to the target DNA molecule. Following hybridization, solid phase PGR can he performed to create a popula tion of discrete amplieons, which can then be sequenced to identify the presence of said molecule

(01021 FIG, 7A shows a top view of a portion of a reaction vessel 700, In articular _s a reaction vessel wall 702 formed from optically transparent aterial is depicted. Aiiinteribr- facing surface of reaction vessel wall 702 i plated with light absorbing layer 704, which has a serpentine or meandering pattern that covers majority of the interior- facing surface of reaction vessel wall 702, Examples of reaction vessel walls are depicted in FIGS. 1 -IB by housing components 102 arid 152 The reaction vessel wall 702 corresponds to that portion of housing components 102 and 152 that harm reaction chamber 104. Because light absorbing layer 704 is positioned upon the interior-facing surface of reaction vessel wall 702. any photonic energy converted into heat energ by light absorbing layer 704 can be conducted directly into solution positioned within a reaction chamber defined at least in part by reaction vessel wall 702. Light absorbing layer 704 is formed from electrically conducti ve material such as elemental gold, chromium, titanium, germanium, nickel, platinum, graphene and silver or a gold alloy such as, e.g., gold-gennanium, gold-chromium, gold-titanium, gold- chromium-gerraaniuro and gold-titanium-germanium, combinations thereof, or the like. [0103! As depicted in FIG. 7A, the pattern of light absorbing layer 704 can be arranged so as to form ail electrically eondoctive pathway that travels across the majority of an area provided by the interior-facing surface of reaction vessel wail 702. The large are occupied by light absorbing layer 704 allows _: light absorbing layer 704 to absorb a significant amount, for examp le , a maj ority of the photonic en ergy directed at reaction vessel wall 702. Close up view 706 shows how adjacent segments 70S of light absorhing layer 704 pan be at least twice as wide (w) as a gap (d) 710 separating the adjacent segments. In some embodiments, adjacent segments oflight absorbing layer 704 can be up to ten times as wide as gap 710. By including only a narrow gap 710 between adjacent segments as depicted, light absorbing layer 704 can have a density sufficient to efficiently heat material within reaction vessel 700

[0104! hr some embodiments, light absorbing: layer 704 can cover between 5% and 95% of the surface area of reaction vessel wall 702. When larger amounts of heat transfer are required, light absorbing layer 704 can cover between 50% an 95% of the surface area reaction vessel wall 702. In some embodiments, gap 710 can be less than or equal to 800 nro. This small gap size has the benefit of filtering out some infrared wavelengths of light from entering reaction vessel 700 while simultaneously allowing light having a shorter wavelength, for example, visible or ultraviolet wavelengths, to pass through the small gaps between the adjacent segments. An additional benefit of this configuratio is that longer wavelengths of light (i.e. wavelengths longer than 800 tun) that are associated with light waves imparted by a photonic energ source are in most cases too large to pass through the gap an therefore unable to bypass the light absorbin layer. In this way, the small gaps between adjacent segments do not materially degrade the conversion of photonic energy into beat energy.

J 0105] FIG, 7 A also shows how electricity can be routed through the electrically conductive pathway defined by light absorbing layer 704. In particular, a first electrical pad 712 is depicted at a first side of reaction vessel 700 and electrically couples a first side of light absorbing layer 704 to an electrical input (In), e,g„ coupled to a first terminal of an electrical energy source such as a DC source, an AC source, a battery, etc. A second electrical pad 714 is depicted at a second side of reaction ves sel 700 and electrically couples a second side of light absorbing layer 704 to an electrical output (Out), e.g., coupled a second terminal of the electrical energy source. By measuring a voltage change (e.g., a voltage drop) between the first and second electrical pads, the electrical resistance of light absorbing layer 704 can be determined. As described herein, this electrical resistance can be correlated with

5 temperature and may therefore be used as a proxy to determine temperature in a reaction vessel or chamber.

(01061 Since the materia! making up light absorbing layer 704 is an electrically conductive material, electrical resistivity will generally increase with increasing temperatures. For example, an electrical resistivity of copper and gold generally increases linearly with respect:0 to increases in temperature while other electrical conductors have non-linear responses to increases in temperature, These predictable changes in electrical resistance due to

temperature allows for accurate measurements of temperature to be made within reaction vessel 700 without the need for a separate te perature sensor. For example, a resistance of the light absorbin layer 704 at a given time can be determined by measuring a voltage 5 change when electricity is passe through the light absorbing layer 704, In this example, an associated processor may determine a temperature corresponding to _: the determined resistance (e.g., by accessing a lookup table or based on a function that calculates temperature based on resistance). This method of detenniniug temperature allows light absorbing layer 704 to act to both efficiently add heat to reaction vessel 700 and to measure how quickly that heat0 increases a temperature of the interior of reaction vessel 700. It should be n oted that the

changes in electrical resistance of the light absorbing layer due to: temperature change are: caused b small changes in the latice structure of the metal resulting from the changes in temperature. In some embodiments, changes of the electrical resistivity of the light absorbing layer over time can also be used to: measure a structural integrity of the light absorbing layer.5 Periodic calibration tests can be performed to identify these changes over time.

( 01071 FIG. 7B show's how light absorbing layer 704 can be arranged in a different pattern than the one depicted in FIG. 7A. In particular, the pattern illustrated in FIG, 7B forms an electrically conductive pathway that sequentially covers each of fou quadrants of reaction vessel 700. The multiple turns of the electrically conductive path help increase an electrical0: resistance of the electrically conducti ve path, thereby ^: helping make changes hi the resistance more noticeable to a processor monitoring the voltage different between electrical pads 712 and 714. This configuration also shows how the electrically conductive path way formed by light absorbing layer 704 can enter and exit reaction vessel 700 in substantially the same area. The entrance point through which the electrically conductive pathway enters and exits reaction vessel 700 can coincide with a liquid entry channel or can be used solely for the entr and exit of the electrical ly conductive pathway formed by light absorbing layer 704. FIG. 7B also shows how a central portion 716 of light absorbing layer 704 can he entirely 5 disassociated from the electrically conductive pathway and function primarily as a light absorbing layer to transfer heat converted from photonic energy into solution within reaction vessel 700.

{010$] FIG. 7C shoves a third configuration of light absorbing _: layer 704 in which a central region 718 of light absorbing layer 704 defines an electrically conductive pathway coupled to:0 electrical pads 712 and 714 and ¾ peripheral region 720 of light absorbing layer 704 is not electrically connected to the central regio and is used only for heat generation and transfer. This configuration results in temperature measurements made using die electrically conductive pathway being biased toward the central region of reaction vessel 700, This may be helpful where reactions are more likely or expected to occur in particular regions of a 5 reaction vessel, and a peripheral region of th reaction vessel is expecte to be at a slightly different temperature than a central region. For example, this might be the case tor a reaction vessel in which the peripheral region is shallower than the central region, as in the example reaction vessels 100 and 150 of FIGS. 1 A~1B, which have a curved or graded reaction chamber 104. As another example, a concentration of bound molecules (e.g., in solid-phaseQ PGR, as depicted, for example in FIGS . 6A-6D) may be higher in particular regions of a reaction chamber (e. , referencing FIGS , 6A--6D, the discrete regions 604 of reaction chamber 609 of reaction vessel 600).

[0109] FIG. 7D shows a fourth configuration of light absorbing layer 704 in which the electrically conductive pathway formed by light absorbing layer 704 is concentrated in5 multiple discrete regions 722, 724, and 726 across the interior- lacing surface of reaction vessel wall 702. In this embodiment, it central region 730 of light absorbing layer 704 functions only to generate and transfer heat into reaction vessel 700. This configuration can be beneficial as temperature determinations made by measuring the voltage change across electrical pads 712 and 714 can provide a localized temperature determination specific to0 discrete: regions 722, 724, and 726. This can be particularly beneficial where reactions are localized to these particular discrete regions 722, 724, or 726 as energy changes resulting from these reactions can have some localized effect upon temperature of the reaction vessel. FIGS: 6A-6D show examples of specific reactions in which target molecules are affixed to specific locations within a reaction chamber such that reactions occur in predictable location within the reactions chamber. In these types of exemplary reactions patterned thin fi lms can he positioned in the specific locations to allow for more accurate monitoring of the reactions, j O! lO j In some embodiments, the central region 730 may not include a light absorbing layer. In these embodiments, only the discret regions 722, 724, and 726 may he include light absorbing layers in these embodiments. These embodiments may operate in a manner similar to the example i llustrated in previous figures (e.g ₅ the discrete regions 722, 724, and 726 may ^¬ be analogous to the discrete regions 302, 304, 306, or the discrete regions 604:). ¾ som of these embodiments, each of these di screte regions may be coupled to electrical pads (e.g., similar to die electrical pads 712 and 714 in FIG 7,4) for individuall measuring voltage changes an thereb individually c alcu luting temperature at or near each of the discrete regions. In other embodiments, two or more of the discrete regions may he grouped together such that a single voltage change is measured for the grouped discrete regions. For example, a first voltage change may be measured across two discrete regions of a first set (e.g., with a first set of electrical pads), and a second ¹ voltage change may be measured across three discrete regions of a second set (e.g., with a second set of electrical pads).

[ill llj FIG. 8 shows a reaction vessel 800 with a light absorbing layer 802 distribute across reaction vessel wall 804. Light absorbing layer 802 includes a first electrically conductive pathway localized in two discrete regions 806 and 807 of light absorbing layer 802. This d lows for determinat on of the temperature of discrete regions 806 and 807 by measuring a voltage change between electrical pads 80S and 810. Light absorbing layer 802 includes a second electrically conductive pathway localized in one discrete region 812 of light absorbing layer 802 and allows for determination of a temperature of reaction vessel 800 within discrete regi on 812 of reac tion vessel 800 by measuring a voltage change across electrical pads 814 and 816. Including two different electrically condnetive pathways allows for detemaination of temperature within different porti ons Of reaction vessel 800. This can be particularly helpful in configurations similar to the one shown in FIG 3B, in which multiple different light sources apply energy to different regions of a reaction vessel.

[0112] Separating the electrically conductive pathways also provides a certain amount of thermal isolation that can allow a larger thermal gradient to be applied. For -example, first and second light sources could be directed at respective discrete regions 806 and 807 and a third light source could be directed at discrete region 812, This woul allow for large differentials in energy input to the three discrete regions and a resulting temperature differential could be tracked due to the: presence of the two discrete electrically conductive pathways. In some embodiments a larger number of discrete electrically condactive pathways coul be utilized to track a larger number of thermal gradients in different regions of a reaction vessel. For example, the depicted first electrically conductive pathway could be split in two in order to track each of discrete regions 806 and 807 separately. Configurations having as many as four, five, or six or more electrically conductive pathways are also possible and deemed to be within the scope of this disclosure.

[0113] In some embodiments, a reaction vessel may include electrically conductive pathways that are separate from light absorbing layers, FIG _* 9A show's -a top view' of a portion of an example reaction vessel 900 ha ving _: light absorbing layer 902 arrange upon a reaetion vessel wall 904. As depicted, light absorbing layers 902 -4 an 902-2 form two different electrically conductive path ways covering different portions of reaetion vessel wall 904 (e.g., the top half and the bottom half of FIG. 9A), Light absorbing layers 9024 and 902- 2 may be formed from a material known to be efficient at converting photonic energy to heat energy. Electrically conductive pathways 906-1 and 906-2 can be disposed, for example, on top of, beneath, or otherwise adjacent to light absorbing layers; 9024 an 902-2. The electrically conductive pathways 906 can be formed from a material known to have predetermined electrical resistance (e.g., material including platinum, gold, nickel, copper) and a predictable increase and decrease in electrical resistance due to changes in temperature. Electrical energy can he routed through electrically conductive pathways 9064 and 906-2 using electrical pads 908-1 and 908-2. As describe more folly below", the embodiment illustrated in FIG, 9A enables functional separation between the material utilized:0s the light absorbing layer to convert photonic energy to heat energy and the material utilized as the electrically conductive pathway. Accordingly, a first material that absorbs heat efficiently, but is highly conductive, can be utilized in conjunction with a second material that is not an efficient heat absorber, but is highly resistive.

[004] FIG. 9B shows a cross-secti onal view of reaction vessel 900 in accordance with section line A*A of FIG. 9 A, Although FIG. 9B only illustrates across section of a segment along the top half of reacti on vessel wall 904, a cross section of a similar segment along the bottom half of the reaction vessel wall 904 may be similar. In particular, FIG 98 shows how electricall conductive pathway 906-1 can be electrically insulated from light absorbing layer 902-1 by an electrically insulating layer 910. In this way, electrical current received at electrical pads 908-1 through electrically conductive pathway 906-1 can be prevented from being unintentionally conducted to light absorbing layer 902- L The electrical isolation provided by embodiments of the present i vention can be important in obtaining accurate voltage change readings that benefit from the higher electrical resistivit of the material used to form an electrically conductive pathway 906-1 , Although FIG. 9B illustrates a particular configuration, with the electrically conductive pathway 906-1 disposed on the electrically insulating layer 910, which is, in turn, disposed on the light absorbing layer 902-1, any suitable configuration is contemplated. For example, the electrically conductive pathway 906- 1 may be disposed beneath the electrically insulating layer 910, which may be disposed beneath the light absorbing layer 902-1. in some embodiments, light absorbing layer 9024 may not be electrically conductive, in which case, the electrically insulating layer 910 may be unnecessary. For example, in these embodiments, electrically conductive pathway 906- 1 may be disposed directly on top of, beneath, or adjacent to light absorbing layer 902- 1. In some embodiments, electricall conductive pathway 906-1 can be substantially narrower than the segments of light absorbing layer 902-1 upon which it is positioned. This narrow

configuration ofe!ectricaliy conductive pathway 906-1 allows portions of light absorbing layer 902- Ϊ to remai exposed so that heat generate within lightabsorbing layer 902-1 can be conducted directly into solution flowing over and around light absorbing layer 902-1.

10115 _. 1 As mentioned above with respect to the discussio related to FIG. 7A, it may be advantageous in: some: cases to have a higher density of the light absorbing layer 704. In some embodiments, a reaction vessel may include oneof more light absorbing layers without gaps _* In these embodiments, the reactio vessel may include one or more electrically conductive: pathways that are separate from the one or more light absorbing layers (similar to the examples discussed above with reference to FIGS, 9A-9B), For example, a reaction vessel may include one or more serpentine electrically conductive pathways similar to those illustrated in FIG, 9A (e.g„ electrically conductive pathways 906- 1 and 906-2), but may inc lude a single light absorbing laye that spans the entirety of the reactio vessel 900. As another example, a reaction vessel may include one or more serpentine electrically conductive pathways similar to those illustrated in PIG 9A (e.g., electrically conductive pathways 906- 1 and 906-2), but may include two discrete light absorbing regions. In this example, a first discrete light absorbing region may be beneath or atop the electrically conductive pathway 906-1, and a secon discrete light absorbing region may be beneath or ato the electrically conductive pathway 906-2. [0116J FIG. 10 shows a feedback control loop 1000 for modulating output of an energy source (e.g., a photonic energy source) into a reaction vessel. In particular embodiments, the feedback control loop 1000 can receive a desired temperature profile for a reaction chamber of the reaction vessel associated with a particular procedure. For example, a desired reaction chamber temperature 1002 may be determined for a given time. A controller 1006 including: one or more processors may be configured to sen a first energy source input to a first energy source 100S for imparting energy to the reaction vessel in order to increase temperature of the reaction vessel. For example, the: first energ source 3008 may be a photonic energy source {e.g., an LED), A temperature of the reaction chamber can be monitored by a temperature sensor disposed within the reaction vessel. In: so e embodiments, as described herein, the temperature sensor can be incorporated into a light absorbing layer that is responsible for transferring heat from a photonic energy s ource into the reaction chamber. For example, die temperature sensor may measure a voltage change following application of electrical energy by a second energy source 1005 (e.g,, an AC source, a battery):, for example, to an electrically conductive pathway (e.g., the light absorbing layer). In some embodiments, the temperature sensor can be a discrete sensor not directly associated with a; light absorbing layer (e.g,, similar to the examples illustrated in FIGS. 9A--9B), The measurements taken by the temperature sensor may be used to derive a temperature of the reaction chamber 1004, which may be subtracted from desired reaction chamber temperature 1002. A controller 1006 may receive the difference between the actual and desired temperature, in some embodiments, the controller 1006 may modulate the photonic energy source input signal transmitted to a first energy source 1008 (e g., a photonic energy source) to bring the reaction vessel to the desired temperature and/or maintain the the reaction vessel at the desired temperature, Controller 1006 can take many forms including the form of a PI, P D, or FID controller. |01171 fi GSi 11A-1.1 C show: different light absorbing layer configurations formed from patterned metallic film that include discrete regions arranged so that a density of the light absorbing layer varies across a surface of a reaction vessel wall. In particular, FIG. 1 LA shows a reaction vessel 1100 having _: a reaction vessel wall 1102 that includes discrete regions of a light absorbing layer 1 104 taking the shape of concentric rings 1 1:06. A spacing between the concentric rings can get incrementally larger as the rings get increasingly closer to a central region of light absorbing laye 1 104. Density profile .1 108 shows an exemplary' light absorbing layer density profile indicating how the density of the: metallic film making up: fight absorbing layer can be substantially greater along its peripheral region than in its central region. In this way, a periphery of the light absorbing layer can be more effici ent at absorbing and transferring heat into reaction vessel 1100 than a centra! portion of the light absorbing layer 1 104, In some embodiments, this configuration can be beneficial where a light source being directed at the light absorbing layer is more intense at its center than along its periphery. In such a case, reducing the density of light absorbing layer 1104 in the central region and increasing the density' of light absorbing layer 1104 it along the periphery can help to normalize an amount of energy introduced across the surface of reaction vessel wall I t 02 upo which light absorbing layer 1104 is disposed. This may help make heating uniform across the reaction vessel 1100.

[01181 FIG. 1 IB shows a cross-sectional side view of reaction vessel 1100 in accordance with section line A- A of FIG. ! IA. FIG. 1 IB shows a thickness of the patterned metallic film by illiistratiitg a thickness of the concentric rings 1 lObibrming the light absorbing laye 1 104, FIG. 1 IB also depicts how a shape of reaction vessel wall 1 102 can be substantially flat. This differs fro the reaction vessels shown in FIGS. 1 A- 1 B which show concave or graded geometries for the reaction vessefwall, in some embodiments, die concave or graded geometry·' can perform a similar function as the density gradient of the light absorbing layer by reducing the amount of solution posi tioned along the periphery of the reaction vessel thereby reducing the amount of heat needing to be sent to the periphery and helping to establish a relatively uniform distribution of heat within reaction vessel 1100. In some embodiments, a density gradient can; be used In combination with a sligh tly concave geometry for a reaction vessel wall to achieve an even distribution of heat within the reaction vessel.

[0119| FIG. 11 C shows an alternative variable density light absorbing layer configuration for a reaction vessel 1150 in which hexagonal discrete regions 1 152 of a light absorbing layer take the form of Hexagonal discrete regions. At the enter periphery of the light absorbing layer the hexagonal discrete regions 1152 are only separated very slightly. The hexagonal discrete regions 1152 are then separated by incrementally greater distance towards the central region of a reaction vessel wall. A size of the discrete hexagonal regions can also be reduced towards the central region to help facilitate the larger gaps between the discrete regions. It should be appreciated that while the hexagonal shape does facilitate an efficient spacing of the discrete regions, other shapes for the discrete regions are possible and deemed to be within the scope of the disclosure. For example, rectangular, triangular, or even curved non-polygonal shapes are possible FIG 1 i C show's how a geometry of the reaction vessel itself can have a non-circular / non -rectangular geometry well suited for accommodating a size and shape of the discrete regions of the light absor bing layer. Furthermore _; re action vessels in general can have other shapes and sizes as neede to help facilitate uniform distribution of heat within tire reaction vessel.

[01201 FIG. 11D-11E show exemplary side views of a distri bution of heat wi thin reacti on vessels using conventional light absorbing layers ami patterned light absorbing layers. FIG. H D shows an interior portion of a reaction vessel 1160 including light absorbing layers 1162 on interior- facing surfaces of both an upper reaction vessel wall 1164 and: a lower reaction vessel wall 1164 of reaction vessel 1160. Li ght absorbing layers 1 162 can take the form of solid layers of metallic _: film, FIG. 1 ID also shows how when energy sources 1168 illuminate light absorbing layers 1162 an uneven distribution of heat builds up within reaction vessel 1160. This uneven distribution of heat occurs because heat is injected substantially evenly across upper and lower reaction vessel walls i 164 and 1166 of reaction vessel 1160 and the heat being added is only able to escape reaction vessel 1160 through lateral walls i 170 of reaction vessel 1 160. This results in central region 1 172 being the warmest region and the dot patterns indicate how the heat gradually drops until reaching its _. lowest temperature in peripheral region 1174 of reaction vessel 1160 due to the dissipation of the heat through lateral walls 1 170- This highly varied distribution of beat can be undesirable where a more uniform distribution of heat is desired.

[01211 FIG. 1 IE show's how- patterned light absorbing layers 1 186 can be adhered to Upper and lower reaction vessel walls 1164 and 1 16 of reaction vessel 1 180. Patterned light absorbing layers 1182 can have a varied density that results in a larger amount of heat being applied to peripheral regions 1.182 than to a central region 1 184. For example, there ay be a higher density of discrete regions of the light absolving layer 1186 at the peripheral regions 1182 than: at the central region 1184. In some embodiments, a similar result may be effecte by varying the properties of the light absorbing layer 1186 across the reaction vessel 1 180, For example, the composition: of the light absorbin layer 1186 may be varied across the reaction vessel (e.g., discrete regions of the light absorbing layer 1 186 along the peripheral regions 1 182 may be composed of material that absorbs more light energy than discrete regions along the central _: region 1184), As another example, the: thickness of the discrete regions of the light absorbing layer ! 186 may be varied (e.g., discrete regions of the light absorbing layer 1 186 may be thicker along the peripheral regions 1 182). This configuration of light absorbing layers 1182 can be similar to or the satire as the light-absorbing layer configurations depicted in FIGS. 1 1 A an 1 1 C. The use of this varied density light absorbing layer configuration results in heat transferring out of the peripheral region 1182 in t o different directions. A fi rst portion of the heat is conducted into central region 1 184 and a second portion of the heat escapes the reaction vessel through lateral walls 1 170 of reaction vessel 1180. In this way, a thermal gradient within reaction vessel can be normalized even though heat still escapes reaction vessel 1 180 through lateral wails 11 0. While· FIG. 1 11 does depict a slight thermal gradient between centra! region 1184 and peripheral region 1182, it should be noted that in some embodiments, this configuration or a similar one can result in little to no variation in temperature within reaction vessel 1180. [Q1221 FIG _* 12 shows an example method 1200 for operating a reactio vessel assembly.

The method may include, at step 1210, directing light from a light source through at least a portion of the: housing; co ponent at a portion of the light absorbing layer to cause the light absorbing layer to convert the light into heat energy, wherein the light absorbing layer comprises a first electrically conductive pathway. At step 1220, the method may include directing, by an electrical energ source, electrical energy through the first electrically conductive pathway. In some embodiments, the electrical energy is directed through an entirety of the light absorbing layer. At step 1230, the method may include measuring a voltage change across the first electrically conductive pathway. At step 1240, the method may include determining a first temperature within the reaction chamber based upon a voltage change in the electrical energy after passing through the first electrically·' conductive pathway, 1» some embodiments, the method may further include a calibration step for more accurately correlating temperatures to voltage changes so that accurate temperatures for the reaction chamber may be determined. For example, the reaction chamber may be brought to one or more known temperatures (e,g,, as independently measured using separate temperature sensors) and voltages may be measured. The measured temperatures and Voltages may be used to, for example, create a lookup table or otherwise generate a function for determining temperatures within the reaction chamber from a measured voltage. In some embodiments, the method may further include determining that the first temperature is equal to or greater than a desired temperature; and in response to said determination, causing the light source to stop directing light at, or decrease an amount of light directed at, the: portion: of the light absorbing layer. For example, the processor may determine that the first temperature is equal to or greater than the desired temperature. In response, the processor may cause the light source to stop directing ligh at the portion of the light absorbing layer. Alternatively, the processor may decrease n amount of light directed at the portion of the light absorbing layer (e.g., by decreasing power supplied to the light source}- As another example, th processor may determine that the first temperature is less than the desired temperature, and in response may increase an amount of light directe at tire portion of the light absorbing layer. [0123! hi some embodiments, the light absorbing layer may be disposed along the surface of the housing component at a variable density, In some embodiments, the ligh absorbing layer is disposed at a relatively high density along a peripheral portion of the surface of the housing component, and at a relativel low density along a central portion of the surface of the housing component. In some embodiments, the light absorbing layer is disposed at a relati vely low density along a peripheral portion of the surface of the housing component, and at a relati vely high density along a central portion of the surface o f the housing component.

[01 41 P articular embodiments may repeat one or more s teps of the metho of F IG . 12, where appropriate. Although this disclosure describes and il lustrates particular steps of the method of FIG. 12 as occurring in a particular order, this disclosure contemplates any suitable steps of the method of FIG . 12 occurring in any suitable order. Moreover, although this disclosure describes and illustrates an example method for operating a reaction vessel assembly, including the particular steps of the method of FIG. 12, this disclosure

contemplates any suitable method for operating a reaction vessel assembly, including any sui table steps, which may include ail, some, or none of the steps of the method of FI G 12, where appropriate. Furthermor , although thi disclosure describes and illustrates particular components, devices, or systems carrying out particular steps of the method of FIG. 12, this disclosure contemplates any suitable combination of any suitable components, devices, or systems carrying out any suitable steps o f the method of FIG. 12,

[0125| The foregoing description, for purposes of explanation, used specific nomenclature to provi de a thorough understand ing of the described embodi ents. However, it will be apparent to one skilled in the art that the specific details are not required in order to practice the described embodiments. Thus, the foregoing descriptions of specific embodiments are presented for purposes ¾f illustration and descripti on. They are not intended to be exhaustive or to limit the described embodiments to the precise forms disclosed. It will be apparent to one of ordinary skill in the art that many modificati ons and variations are possibl e in view of the above teachings.