Nanofabrication of deterministic diagnostic devices

Abstract

We provide diagnostic equipment. [Solution] A diagnostic chip for detecting biomarkers and trace nanoparticles in chemical mixtures or water. The diagnostic chip comprises one or more input sections into which a sample containing particles of different sizes is introduced. Furthermore, the diagnostic chip comprises multiple separation regions into which the sample is pressurized as it passes through. Each separation region comprises a deterministic transverse displacement array, and the deterministic transverse displacement arrays in two or more of these separation regions have different etching depth profiles. In this way, the diagnostic chip effectively detects biomarkers and trace nanoparticles in chemical mixtures or water.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 058,284, “Nanofabrication of Point-of-Use Deterministic Diagnostic Devices,” filed on 29 July 2020, which is incorporated herein by reference in its entirety.

[0002] The present invention relates in general to diagnostic devices, and more particularly to nanofabrication of deterministic diagnostic devices. [Background technology]

[0003] Diagnostic devices, such as medical diagnostic equipment, help clinicians measure and observe various aspects of a patient's health to form a diagnosis. Once a diagnosis is made, the clinician can then prescribe an appropriate treatment plan.

[0004] Medical diagnostic equipment is found in outpatient treatment centers for adults and pediatrics, in emergency rooms, and in hospital wards and intensive care units.

[0005] Such diagnostic devices can be used to detect low concentrations of biomolecules, providing early disease detection and monitoring patient response to treatment. These diagnostic tools can help clinicians make critical decisions regarding treatment methods and improve patient outcomes. In the early stages of a disease, the concentrations of disease markers are very low and difficult to detect in typical media such as blood, urine, plasma, and serum. By capturing and isolating biomarkers such as tumor cells and exosomes, sensors can make it possible to detect them. In a biomedical context, a biomarker or biological marker is a measurable indicator of some biological state or condition. Similarly, there are important applications in detecting trace amounts of nanoparticles in chemical mixtures or water.

[0006] Unfortunately, there are currently no diagnostic devices that can effectively detect such biomarkers, or any means to effectively detect trace amounts of nanoparticles in chemical mixtures or water. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International application No. PCT / US2018 / 060176 [Non-patent literature]

[0008] [Non-Patent Document 1] Huang et al., "Continuous Particle Separation Through Deterministic Lateral Displacement," Science, Vol. 304, No. 5673, May 2004, pp. 987-990. [Non-Patent Document 2] McGrath et al., "Deterministic Lateral Displacement for Particle Separation: A Review," Lab on a Chip, Vol. 14, No. 21, 2014, pp. 4139-4158. [Non-Patent Document 3] Inglis et al., "Critical Particle Size for Fractionation by Deterministic Lateral Displacement," Lab on a Chip, Vol. 6, No. 5, May 2006, pp. 655-658. [Non-Patent Document 4] Wunsch, "Nanoscale Lateral Displacement Arrays for the Separation of Exosomes and Colloids Down to 20 nm", Nature Nanotechnology, Vol. 11, No. 11, November 2016, pp. 936-940. [Non-licensed Document 5] Cherala, "Nanoshape Imprint Lithography for Fabrication of Nanowire Ultracapacitors", IEEE Transactions on Nanotechnology, Vol. 15, No. 1, January 2016, pp. 448-456 [Non-licensed Document 6] Mallavarapu, "Enabling Ultra-High Aspect Ratio Silicon Nanowires Using Precise Experiments for Detecting Onset of Collapse", Nano Letters, Vol. 20, No. 11, 2020, pages 7896~7905 [Non-licensed Document 7] Mallavarapuら, "Scalable Fabrication and Metrology of Silicon Nanowire Arrays made by Metal Assisted Chemical Etching", IEEE Transactions on Nanotechnology, Vol. 20, 2021, pages 83~91 [Non-licensed Document 8] Sharma, "SERS: Materials, Applications and the Future", Materials Today, Vol. 15, Nos. 1-2, January-February 2012, pp. 16-25 [Non-licensed Document 9] Nichkalo et al., “Silicon Nanostructures Produced by Modified MacEtch Method for Antireflective Si Surface,” Nanoscale Research Letters, Vol. 12, No. 106, 2017, pp. 1-6. [Non-Patent Document 10] Choi et al., "UV Nanoimprint Lithography," Handbook of Nanofabrication, edited by Gary Wiederrechts, Elsevier Press, October 2009, pp. 310, pp. 149-181. [Non-Patent Document 11] TA Green, “Gold Etching for Microfabrication”, Gold Bulletin, Vol. 47, No. 3, 2014, pp. 205-216 [Non-Patent Document 12] Volker Lehmann, "Electrochemistry of Silicon: Instrumentation, Science, Materials and Applications", Wiley-VCH Verlag GmbH, Weinheim, 2002, pp. 1-115. [Non-Patent Document 13] Alexey Ivanov, "Silicon Anodization as a Structuring Technique: Literature Review, Modeling and Experiments", 2018, pp. 1-316 [Overview of the Initiative] [Means for solving the problem]

[0009] In one embodiment of the present invention, a diagnostic chip includes one or more input portions, and a sample containing particles of different sizes is introduced into at least one of the one or more input portions. The diagnostic chip further includes a plurality of separation regions, the sample is pressurized when passing through the plurality of separation regions, each of the plurality of separation regions includes a deterministic lateral displacement array, and the deterministic lateral displacement arrays in two or more of the plurality of separation regions have different etching depth profiles.

[0010] In another embodiment of the present invention, an apparatus for separating one or more biological species includes a separation region including a microscale or nanoscale structure, and a substrate under the separation region is non-porous. The apparatus further includes at least one output region, and a substrate under the at least one output region is porous.

[0011] As can be better understood from the following detailed description of the present invention, the above has rather generally outlined the features and technical advantages of one or more embodiments of the present invention. Additional features and advantages of the present invention that can form the subject matter of the claims of the present invention will be described hereinafter.

[0012] A better understanding of the present invention can be obtained by considering the following detailed description in conjunction with the following drawings.

Brief Description of the Drawings

[0013] [Figure 1] FIG. shows silicon nanopillars fabricated by catalytically influenced chemical etching (CICE) for deterministic lateral displacement (DLD)-based particle separation according to an embodiment of the present invention. [Figure 2] FIG. shows an apparatus (a "desktop" apparatus) for providing liquid and gas to a diagnostic chip (a "disposable chip") and inspecting the diagnostic chip according to an embodiment of the present invention. [Figure 3A] FIG. shows an embodiment of a disposable diagnostic chip according to an embodiment of the present invention. [Figure 3B] FIGs. show an embodiment of a disposable diagnostic chip according to an embodiment of the present invention. [Figure 3C] This figure shows one embodiment of a disposable diagnostic chip according to one embodiment of the present invention. [Figure 3D] This figure shows one embodiment of a disposable diagnostic chip according to one embodiment of the present invention. [Figure 4A] This figure shows a second embodiment of a disposable diagnostic chip according to one embodiment of the present invention. [Figure 4B] This figure shows a second embodiment of a disposable diagnostic chip according to one embodiment of the present invention. [Figure 5A] This is a top view of a pillar array according to one embodiment of the present invention. [Figure 5B] This figure shows three arrangements of a pillar array according to one embodiment of the present invention. [Figure 6] This figure shows one embodiment of a diagnostic chip according to one embodiment of the present invention, in which micro / nanofabricated silicon is integrated with a top transparent substrate, and a micro / nanopillar array acts as a spacer that creates a microscale gap between the bottom and top substrates of the pillars. [Figure 7] This is a flowchart of a method for manufacturing silicon nanopillars according to one embodiment of the present invention. [Figure 8A] This is a cross-sectional view of a silicon nanopillar being manufactured using the steps shown in Figure 7 according to one embodiment of the present invention. [Figure 8B] This is a cross-sectional view of a silicon nanopillar being manufactured using the steps shown in Figure 7 according to one embodiment of the present invention. [Figure 8C] This is a cross-sectional view of a silicon nanopillar being manufactured using the steps shown in Figure 7 according to one embodiment of the present invention. [Figure 8D] This is a cross-sectional view of a silicon nanopillar being manufactured using the steps shown in Figure 7 according to one embodiment of the present invention. [Figure 9A] This figure shows an image of a 4-inch wafer after the process steps shown in Figure 8A, according to one embodiment of the present invention. [Figure 9B]This figure shows an image of a 4-inch wafer after the process steps shown in Figure 8B, according to one embodiment of the present invention. [Figure 9C] This figure shows an image of a 4-inch wafer after the process steps shown in Figure 8C, according to one embodiment of the present invention. [Figure 9D] This figure shows an image of a 4-inch wafer after the process steps shown in Figure 8D, according to one embodiment of the present invention. [Figure 10] This figure shows a top-down scanning electron microscope (SEM) image of silicon nanowires fabricated by metal-assisted chemical etching (MACE) according to one embodiment of the present invention. [Figure 11] This figure shows a cross-sectional SEM image of a silicon nanowire fabricated by MACE according to one embodiment of the present invention. [Figure 12] This figure shows an exemplary side barrier array for particle separation according to one embodiment of the present invention. [Figure 13] This is a flowchart of a method for fabricating a self-aligned pillar using a MACE process according to one embodiment of the present invention. [Figure 14A] This is a cross-sectional view showing the fabrication of a self-aligned pillar using the MACE process, using the steps described in Figure 13 according to one embodiment of the present invention. [Figure 14B] This is a cross-sectional view showing the fabrication of a self-aligned pillar using the MACE process, using the steps described in Figure 13 according to one embodiment of the present invention. [Figure 14C] This is a cross-sectional view showing the fabrication of a self-aligned pillar using the MACE process, using the steps described in Figure 13 according to one embodiment of the present invention. [Modes for carrying out the invention]

[0014] As mentioned in the background section, there are currently no means for diagnostic devices to effectively detect biomarkers or trace amounts of nanoparticles in chemical mixtures or water.

[0015] The principle of the present invention provides means for effectively detecting biomarkers and effectively detecting trace amounts of nanoparticles in chemical mixtures or water.

[0016] In one embodiment, the principle of the present invention performs such detection using a technique referred herein as “deterministic transverse displacement (DLD)”. DLD is a microfluidic technique that separates particles in a fluid medium based on their size using a specific arrangement of an array of pillars placed within a microfluidic channel. The separation mechanism is determined by the gap between the pillars and the arrangement of the pillars. For further explanations of DLD, see Huang et al., "Continuous Particle Separation Through Deterministic Lateral Displacement," Science, Vol. 304, No. 5673, May 2004, pp. 987-990; McGrath et al., "Deterministic Lateral Displacement for Particle Separation: A Review," Lab on a Chip, Vol. 14, No. 21, 2014, pp. 4139-4158; Inglis et al., "Critical Particle Size for Fractionation by Deterministic Lateral Displacement," Lab on a Chip, Vol. 6, No. 5, May 2006, pp. 655-658; and Wunsch et al., "Nanoscale Lateral Displacement Arrays for the Separation of Exosomes and Colloids Down to 20 nm," Nature Nanotechnology, Vol. 11, No. 11, November 2016, can be found on pages 936-940, and each of these is incorporated herein by reference in its entirety.

[0017] Referring to the drawings in detail, Figure 1 shows a silicon nanopillar fabricated by catalytic chemical etching (CICE) for DLD-based particle separation according to one embodiment of the present invention.

[0018] As shown in Figure 1, the pillar array 101 required for DLD receives a sample containing a mixture of particles of multiple sizes and shapes via an inlet 102 and generates multiple streams of particles separated by size and / or shape via an output stream 103. In one embodiment, the DLD pillar array 101 generates a pattern that maximizes separation efficiency and throughput using the following variables: pillar size and spacing, pillar shape (e.g., circular, triangular, rhombic, streamlined, etc.), pillar array arrangement and skew angle, and pillar height before collapse. Furthermore, as shown in Figure 1, Figure 104 of the sample in the inlet 102 corresponds to pillars with a height of 2 micrometers and a spacing of 30 nm, fabricated by a ruthenium-catalyzed CICE. In addition, as shown in Figure 1, Figure 105 of the outlet stream 103 includes silicon (Si) pillars with a height of 4 micrometers and a spacing of 30 nm, fabricated by a gold-catalyzed CICE. Furthermore, as shown in Figure 1, Figure 106 of the DLD pillar array 101 includes silicon (Si) nanopillars with a rhombic cross-section.

[0019] In one embodiment, the DLD pillar array 101 is manufactured using nanolithography, such as nanoimprint lithography combined with a metal-assisted chemical etching (MACE) process. Further details regarding manufacturing using DLD and MACE can be found in Cherala et al., "Nanoshape Imprint Lithography for Fabrication of Nanowire Ultracapacitors," IEEE Transactions on Nanotechnology, Vol. 15, No. 1, January 2016, pp. 448–456; Mallavarapu et al., "Enabling Ultra-High Aspect Ratio Silicon Nanowires Using Precise Experiments for Detecting Onset of Collapse," Nano Letters, Vol. 20, No. 11, 2020, pp. 7896–7905; and Mallavarapu et al., "Scalable Fabrication and Metrology of Silicon Nanowire Arrays made by Metal Assisted Chemical Etching," IEEE Transactions on Nanotechnology, Vol. 20, 2021, pp. 83–91, each of which is incorporated herein by reference in its entirety.

[0020] Referring now to Figure 2, Figure 2 shows an apparatus ("tabletop" apparatus) that provides liquid and gas to a diagnostic chip ("disposable chip") according to one embodiment of the present invention and also inspects the diagnostic chip.

[0021] As shown in Figure 2, the desktop devices 201A to 201D have various inputs connected to the disposable diagnostic chip 202 (I1, I2, I3, I, respectively). SThe invention provides (marked as ). Apparatuses 201A to 201D can be referred to as apparatus 201 collectively or individually. Figure 2 shows four apparatuses 201, but it should be noted that the principle of the present invention can utilize any number of desktop apparatuses 201.

[0022] Referring again to Figure 2, if the tip 202 is positioned with sufficient precision on the tip holder 203, which is connected to the main body of the device by a frame, the tip 202 can align with various inlets and receive buffers (such as purified water), pressure sources, solvents required during the operation of the tip 202, etc. The tip 202 also receives a “sample,” which may be the patient’s blood, urine, saliva, serum, etc. In one embodiment, the system is designed to prevent the “sample” from flowing back into any of the reservoirs that hold clean liquids within the device. Further description of the disposable diagnostic tip 202 is provided below.

[0023] Furthermore, as shown in Figure 2, “SZ” corresponds to a sensor zone 204 that is optically inspected using an instrument 205 marked “M / S”, which may be a microscope, fluorescence microscope, spectrometer, Raman spectrometer, etc.

[0024] Referring now to Figures 3A to 3D, Figures 3A to 3D show one embodiment of a disposable diagnostic chip 202 according to one embodiment of the present invention.

[0025] Figure 3A shows a top view of the diagnostic chip, and Figure 3B shows a cross-section along the vertical YY direction shown in Figure 3A. Various input sections (I1, I2, I3, I SA section marked as is shown and represents the same input section as shown in Figure 2. Although only four input sections are shown, these devices may include any number of input sections, including 25 or more. In one embodiment, a “sample” containing particles of different sizes is introduced into one of the input sections I1, I2, or I3. The sample is pressurized together with other liquids such as a buffer solution, and these pass through regions 1 to 4 (301A to 301D, respectively) (identified as “R1”, “R2”, “R3”, and “R4”, respectively). Regions 301A to 301D can be referred to collectively or individually as multiple regions (or “multiple isolation regions”) 301 or region (or “isolation region”) 301, respectively. Again, although only four regions are shown, it should be noted that there may be any number of regions, including 25 or more. In one embodiment, these regions are designed to perform hierarchical filtration of particles leading to each output reservoir (O1 to O3 and output MZ) (identified as outputs 302A to 302D, respectively) where the particle size decreases monotonically. Outputs O4 and 302E collect the remaining liquid and other debris of very small size (e.g., <10 nm or <25 nm). Outputs 302A to 302E can be referred to as multiple outputs 302 or outputs 302, collectively or individually. A sample flowing through region R1 to output O1 is identified as RO1. Similarly, a sample flowing through region R2 to output O2 is identified as RO2. The particle size range reaching O1 to O3, output MZ, and O4 is defined as DLD region R iIt depends on the design. The size, spacing, height, arrangement, orientation relative to the flow direction, and cross-sectional shape of the pillars all contribute to the process. See Huang et al., "Continuous Particle Separation Through Deterministic Lateral Displacement," Science, Vol. 304, No. 5673, May 2004, pp. 987-990; McGrath et al., "Deterministic Lateral Displacement for Particle Separation: A Review," Lab on a Chip, Vol. 14, No. 21, 2014, pp. 4139-4158; Inglis et al., "Critical Particle Size for Fractionation by Deterministic Lateral Displacement," Lab on a Chip, Vol. 6, No. 5, May 2006, pp. 655-658; and Wunsch et al., "Nanoscale Lateral Displacement Arrays for the Separation of Exosomes and Colloids Down The range of particles to be filtered is determined as discussed in "to 20 nm," Nature Nanotechnology, Vol. 11, No. 11, November 2016, pp. 936-940.

[0026] In one embodiment, region 1 is assumed to have a large DLD pillar array with a relatively large diameter (e.g., 25–50 micrometers). Region 2 is assumed to have a slightly smaller DLD pillar array (e.g., in the range of 5–25 micrometers). Region 3 is assumed to have an even smaller DLD pillar array (e.g., in the range of 0.5–5 micrometers). Furthermore, in this design, region 4 is assumed to have the smallest DLD pillar array (e.g., in the range of 25 nm–500 nm). In one embodiment, the spacing between these pillars can be increased to make them "sparse" (as shown in Figure 5B, which will be discussed further below). In one embodiment, the "sparse" pillars have a diameter-to-pitch ratio of 1% to 35% (d / p = 0.01 to 0.35). In one embodiment, the "medium" pillars have a diameter-to-pitch ratio of 35% to 65% (d / p = 0.35 to 0.65). In one embodiment, “dense” pillars have a diameter-to-pitch ratio (d / p = 0.65 to 0.99) of 65% to 99%. In one embodiment, a combination of nanoimprint and MACE is used to achieve the fabrication of these dense pillars, particularly when the spacing between pillars is well below 25 nm. Such fabrications are discussed in Mallavarapu et al., “Enabling Ultra-High Aspect Ratio Silicon Nanowires Using Precise Experiments for Detecting Onset of Collapse,” Nano Letters, Vol. 20, No. 11, 2020, pp. 7896-7905, and in Mallavarapu et al., “Scalable Fabrication and Metrology of Silicon Nanowire Arrays made by Metal Assisted Chemical Etching,” IEEE Transactions on Nanotechnology, Vol. 20, 2021, pp. 83-91.

[0027] In one embodiment, input section I SThis is an optional input for a solvent or chemical substance to be mixed with one of the outputs (in Figures 3A-3B, this is output MZ, which corresponds to the mixing zone). In one embodiment, the output arriving at MZ may be exosomes or antibodies in the size range of 25 nm to 150 nm. Particles such as exosomes S When exposed to a suitable chemical or solvent that arrives in the MZ from the exosome, this chemical can break down the exosome wall and release the exosome's contents, which are biomolecules (biomarkers) representing the cell from which the exosome originated. Finally, in one embodiment, there is an optional sensor zone 204 (marked SZ in Figures 2 and 3A-3B). Thus, the mixing zone (MZ) is output 302 (for example, O F It may include one of the SZ204s (identified as) and / or SZ204. In one embodiment, the sensor zone 204 captures biomarkers released from exosomes and detects them using instruments such as microscopes, fluorescence microscopes, spectrometers, and Raman spectrometers. In particular, the SZ204 may include surface-enhanced Raman spectroscopy (SERS) patterns fabricated with plasmonic materials such as Au, Ag, or Cu, or with more complex material stacks, as discussed in Sharma et al., "SERS: Materials, Applications and the Future," Material Today, Vol. 15, Nos. 1-2, January-February 2012, pp. 16-25, which is incorporated in whole herein by reference.

[0028] It should be noted that exosomes play a crucial role in regulating cellular activity, and there is evidence that they are used, in particular, for the transport of growth factors, microRNAs (miRNAs), mRNAs, and enzymes. In the context of immunomodulation, exosome secretion acts as a unidirectional delivery medium for miRNAs that can regulate gene expression in target cells. Exosome-based cell-free therapies have been identified as a potential approach for regenerative medicine that does not require stem cell transplantation. Once cellular exosomes are isolated using the apparatus described herein, these vesicles can be analyzed in two ways. First, proteomic analysis can be performed to look for surface markers, such as tetraspanins (CD9, CD63, CD81), adhesion proteins, or cell-specific surface markers (e.g., T cell receptors, CAR-T receptors, major histocompatibility complex (MHC) proteins). These surface markers allow for early identification of exosomes in solution and can provide information about the origin of the vesicles and the potential for intercellular communication and recognition between source and target in their physiological environment. The therapeutic potential of exosomes can be further evaluated by analyzing the contents of the exosomes. In one embodiment, the therapeutic potential of exosomes is assessed by lysing isolated exosomes using an organic solvent such as methanol, and then depositing the contents onto a SERS substrate for protein identification and analysis, or by isolating them for further genetic characterization.

[0029] In one embodiment, it may be necessary to etch different regions to different heights in order to appropriately maintain the aspect ratios of these pillars. For example, if the pillars fabricated in region 4 (R4) have a diameter of 100 nm, the pillars fabricated in region 1 (R1) have a diameter of 25 micrometers, while the etching depth in region 1 can be 25 micrometers, while an etching depth of 1 micrometer may be sufficient in region 4. FIG. 3B shows this variable etching depth for each region that causes a transition with a step from one region to the next. However, such a change in the height of the step may cause problems with fluid flow. For example, at the step between R1 and R2, this step may cause some of the smaller particles that need to proceed to region 2, 3 or 4 to become clogged under the step between R1 and R2. This problem can be addressed by an alternative embodiment shown in FIGS. 4A-4B showing a second embodiment of the disposable diagnostic chip according to one embodiment of the present invention.

[0030] FIG. 4A shows a top view of the diagnostic chip, and FIG. 4B shows a cross-section along the vertical direction Y-Y shown in FIG. 4A. As shown in FIGS. 4A-4B, the transitions (between R1 and R2, which is shown as R 12 as shown, between R2 and R3, which is shown as R 23 as shown, between R3 and R4, which is shown as R 34 as shown) are fabricated to be gradual, with ramps between any two regions. The manufacture of these ramps can be difficult, and an approach to address the challenges of their manufacture will be discussed later in this specification.

[0031] An important issue in a multi-region hierarchical DLD device incorporating both microscale and nanoscale DLD regions is the need to approximately match the flow resistance when the flow branches and moves towards different outputs. For example, different flow resistances (Newton seconds per meter -5 or N.s. / m 5It is desirable that the values ​​(measured at) are within approximately 10 times each other. The flow resistance of a channel is defined by the lateral (width) parameter, the channel depth, and the channel length. If the resistance is too low, it can be increased to bring it closer to the resistance of other paths. This increase can be achieved by using one or more of the following approaches: (i) greatly increasing the length - this can be done efficiently by using a helical flow path (e.g., the channel reference for output O3 in Figure 3A, or a winding flow path without any sharp bends that could cause flow interruption), (ii) adding a region of "dense" pillars where d / p > 0.9 or > 0.95, or (iii) reducing the etching height of the channel in local regions. This last concept is shown in Figures 3C and 3D, both of which are cross section ZZ in Figure 3A. In Figure 3C, the etching depth is constant, which is relatively easy to manufacture. However, in Figure 3D, it is shown that the etching depth can vary in a complex way. If such variations in etching depth can be created, hierarchical fluid systems can be designed to have appropriately matched flow resistances. The variations in etching depth during manufacturing will be discussed further below.

[0032] Referring to Figure 5A, Figure 5A shows a top view of a pillar array 101 (Figure 1) according to one embodiment of the present invention. As shown in Figure 5A, the diameter of the pillars decreases from region R1 to region R4, as shown in Figures 3B and 4B. Furthermore, Figure 5B shows three arrangements of the pillar array according to one embodiment of the present invention. As shown in Figure 5B, the three types of arrangements of the pillar array are dense 501A, medium 501B, and sparse 501C patterns.

[0033] Figure 6 shows one embodiment of a diagnostic chip according to one embodiment of the present invention, in which micro / nanofabricated silicon is integrated with a top transparent substrate 601 (e.g., glass, polydimethylsiloxane (PDMS)), and a micro / nanopillar array (not shown in this figure) acts as a spacer, creating a microscale gap 602 between the bottom of a pillar 603 (e.g., a silicon pillar) and the top substrate 601. Also shown is a plexiglass substrate 604 with optionally machined inlet holes 605 and outlet holes 606. In one embodiment, the plexiglass-silicon-top substrate (604-603-601) sandwich is held together by screws as shown in Figure 6.

[0034] Referring now to Figure 7, Figure 7 is a flowchart of Method 700 for manufacturing silicon nanopillars according to one embodiment of the present invention. Figures 8A to 8D show cross-sectional views for manufacturing silicon nanopillars using the steps described in Figure 7 according to one embodiment of the present invention.

[0035] Referring to Figures 8A to 8D in conjunction with Figure 7, in step 701, as shown in Figure 8A, a thermal oxide 802 is deposited on a substrate 801 such as a silicon wafer (for example, a p-type (100) silicon wafer with a resistance of 1 to 10 ohm-cm). In one embodiment, a thermal oxide 802 with a thickness of 30 to 100 nm is grown on the substrate 801.

[0036] In step 702, as shown in Figure 8A, a thin layer of resist material 803 (e.g., polymer) is deposited on the oxide 802 and then patterned to form resist pillars 804 (circular) like pillars in a deterministic transverse displacement pillar array. In one embodiment, the thickness of the resist material is 10-30 nm. In one embodiment, the resist material is patterned using imprint lithography.

[0037] In step 703, the underlying resist material 803 and the underlying oxide 802 are etched, as shown in Figure 8B. In one embodiment, the underlying 10-30 nm residual resist layer 803 is removed (descammed) by oxygen plasma etching. In one embodiment, the underlying oxide 802 is etched using a short buffered oxide etching (BOE) (e.g., 6:1) that isotropically etches the oxide layer 802, or by using a short BOE immersion following reactive ion etching of the oxide 802.

[0038] In step 704, an optional adhesive layer (not shown in Figures 8A-8D) is deposited, followed by the deposition of a thin film of catalyst 805 as shown in Figure 8C. In one embodiment, an adhesive layer such as titanium (Ti) is deposited on the resist pillar 804 and the remaining oxide 802, followed by the deposition of a thin film of catalyst 805 such as silver, gold, palladium, platinum, and ruthenium. In one embodiment, the adhesive layer has a thickness of 2 nm. In one embodiment, the type of catalyst is a MACE catalyst. In one embodiment, the thickness of the catalyst layer 805 is between 2 nm and 50 nm. In one embodiment, the material of catalyst 805 is gold with a thickness of 10 nm or 4 nm.

[0039] In step 705, the structure of Figure 8C is immersed in the MACE solution as shown in Figure 8D. In one embodiment, the patterned wafer is immersed in a MAC solution of 12.5 moles of HF and 1 mole of H2O2. In one embodiment, the etching can be quenched in the wafer, followed by rinsing with water and drying with an air gun supplying clean dry air (CDA). In one embodiment, catalyst 805 (e.g., gold catalyst) can be optionally removed using Transene® potassium iodide-based gold etchant. The remaining resist can be optionally removed using a short oxygen plasma.

[0040] In one embodiment, using method 700, the pillar 804 is designed to prevent clogging of particles in the sample fluid.

[0041] Figures 9A to 9D show images of a 4-inch wafer after each process step shown in Figures 8A to 8D, respectively, according to one embodiment of the present invention.

[0042] Figure 10 shows a top-down SEM (scanning electron microscope) image of silicon nanowires fabricated by MACE as discussed above in relation to Figures 7 and 8A-8D, according to one embodiment of the present invention. In Figure 10, the scale bar is 1 micrometer.

[0043] Figure 11 shows a cross-sectional SEM image of a silicon nanowire fabricated by MACE as discussed above in relation to Figures 7 and 8A-8D according to one embodiment of the present invention. In Figure 11, the scale bar is 1 micrometer.

[0044] Referring to Figures 7, 8A–8D, 9A–9D, 10, and 11, the above process has nanometer-scale resolution and can be used to fabricate pillars with a diameter of 50 nm or less and a spacing of <5 nm. This process can also simultaneously fabricate small (less than 100 nm) and large (>25 micrometers) pillars on an apparatus area and a large etching area (e.g., a square or circular area having a size or diameter of at least 25 micrometers to millimeters). In one embodiment, such a large etching area is fabricated using a gold catalyst deposited as a thin film with or without Ti (<15 nm) in an optional annealing step, such that the gold film has very fine porosity, thereby allowing the etchant to pass through the finely porous gold to etch the large area. A discussion of porous gold is provided by Nichkalo et al., "Silicon Nanostructures Produced by Modified MacEtch Method for Antireflective Si Surface," Nanoscale Research Letters, Vol. 12, No. 106, 2017, pp. 1-6, which is incorporated herein by reference in its entirety.

[0045] In one embodiment, a porous gold film results in the generation of silicon "nanowhiskers" in regions corresponding to the pore locations on the gold film. These silicon nanowhiskers are optionally removed using techniques such as silicon etching with potassium hydroxide (KOH), or oxidation and etching of nanowhiskers using hydrofluoric acid (HF), where oxidation is performed using an oxygen plasma with an oxidizing agent such as nitric acid, electrochemical anodizing, etc.

[0046] In one embodiment, for nanoimprinting of these features, a template replica is fabricated using an electron beam master having holes in the master and creating pillars in fused silica after imprinting and reactive ion etching. Then, as discussed in Cherala et al., "Nanoshape Imprint Lithography for Fabrication of Nanowire Ultracapacitors," IEEE Transactions on Nanotechnology, Vol. 15, No. 1, January 2016, pp. 448-456, the fused silica master is coated with atomic layer deposition of oxide to fabricate pillars of increased size for a given pitch. The resulting fused silica replica can be used for the nanoimprint, followed by the MACE process shown in Figures 7 and 8A-8D.

[0047] In one embodiment, the controlled etching depth variation shown in Figures 3C and 4B is achieved by using one or more of the following approaches.

[0048] In one approach, local temperature is used to control the etching rate of silicon during the MACE process, as discussed in International Application PCT / US2018 / 060176, which is incorporated herein by reference in its entirety. This allows for an increase in the etching rate in regions where the silicon wafer has a higher temperature, resulting in a stepped etching rate in the transition region from a hotter region to a colder region.

[0049] In another approach, the etching rate in a localized region is controlled by controlling the amount of etchant supplied to each part of the wafer. This idea of ​​creating variations in etching depth using control of etchant transport is included in Figure 3 of Mallavarapu et al., "Enabling Ultra-High Aspect Ratio Silicon Nanowires Using Precise Experiments for Detecting Onset of Collapse," Nano Letters, vol. 20, No. 11, 2020, pp. 7896-7905. One method for creating this etchant flow control is to (i) first use the MACE process shown in Figures 7 and 8A–8D to create a short, uniform etching of silicon nanowires (e.g., an etching depth of 100 nm), then (ii) remove the wafer from the etchant, quench it with water and dry it, then (iii) deposit an inkjet-based UV-curable monomer material (such as acrylate, discussed in Choi et al., "UV Nanoimprint Lithography," Handbook of Nanofabrication, edited by Gary Wiederrecht, Elsevier Press, October 2009, see pp. 310, 149–181) to selectively block a portion of the silicon wafer, then (iv) reinsert the wafer into the MACE etchant, thereby continuing the MACE process in the unblocked areas. The UV-curable material can be inkjet onto any of the following:

[0050] (1) Completely blocked regions where further etching should be stopped (e.g., regions R4, MZ, and SZ where the full etching depth has been reached).

[0051] (2) Partially blocked regions (where monomer inkjet droplets are distributed and UV cured before they completely merge, thus leaving small gaps in the interstitial regions of the droplets, and these gaps define the amount of etchant that will penetrate into the underlying silicon for MACE etching).

[0052] (3) Unblocked areas where monomers have not been inkjet-printed, so that MACE etching can continue without interruption.

[0053] In another embodiment, the DLD pillar array 101 (see Figure 1) may have a dense array (staggered or otherwise) of pillars that act as a barrier against fluid flow and lateral leakage (see Figure 12, discussed below). These barrier arrays are essentially dense pillars, as discussed in Figure 5B, and can be "ultra-dense." "Ultra-dense," as used herein, refers to d / p > 0.9 or > 0.95. The cross-sections of the individual pillars of the barrier array do not need to be circular and symmetrical. For example, they can be asymmetrical. An asymmetrical shape restricts fluid leakage outward from the DLD pillar array 101 but allows fluid injection into the DLD pillar array 101 from the outside, which can also be used to perform in-situ operations on the DLD contents, as shown in Figure 12. Figure 12 shows an exemplary side barrier array for particle separation according to one embodiment of the present invention.

[0054] Referring to Figure 12, Figure 12 shows the DLD pillar array 101 together with the inlet manifold 102 and the outlet manifold 103. In one embodiment, the barrier layer / array 1201 can be manufactured together with the DLD pillar array 101 as discussed above, without requiring any separate manufacturing steps. The width of the side barrier array can range from less than a micrometer to above a millimeter. These barrier arrays have the advantage that, on the timescale of these devices, the barrier does not allow the passage of any relevant particles, and only allows a very small percentage of liquid to pass through and permeate.

[0055] In one embodiment, the principle of the present invention involves creating a porous layer for liquid drainage before surface-enhanced Raman spectroscopy (SERS) detection.

[0056] In one embodiment, a buffer solution containing biological or chemical particles to be detected using the diagnostic device discussed herein can be discharged using a porous silicon layer beneath a gold pattern to enhance SERS detection once detected by SERS. In one embodiment, the porous silicon layer is designed to act as a discharge section for the sample liquid while preventing particles in the fluid from penetrating into the pores in the porous silicon layer. In one embodiment, the porous silicon layer is formed after the SERS "bathtub" has been fabricated using MACE in the SZ region of Figures 2, 3A-3B and 4A-4B. In one embodiment, the SERS "bathtub" is connected to a desired DLD array outlet and has an area of ​​2 mm × 2 mm and a depth of 1 micrometer. The "bathtub" is etched together with the DLD array, inlet, and outlet. The gold catalyst is etched away using wet etching (such as potassium iodide-based or aqua regia), plasma etching, or atomic layer etching (as discussed in TA Green, "Gold Etching for Microfabrication," Gold Bulletin, Vol. 47, No. 3, 2014, pp. 205-216, which is incorporated herein by reference in its entirety). In one embodiment, an inkjet is used to distribute a polymer blocking material in all areas except the SERS "bath" region. In one embodiment, a porous layer is fabricated by electrochemical etching of silicon in the SERS "bath" region using an electrolyte consisting of an electric field and HF.In one embodiment, the morphology of the porous layer (porosity, pore size, pore orientation) is controlled by changing the voltage and / or current density across the wafer, as discussed in Volker Lehmann, "Electrochemistry of Silicon: Instrumentation, Science, Materials and Applications," Wiley-VCH Verlag GmbH, Weinheim, 2002, pp. 1–115, and Alexey Ivanov, "Silicon Anodization as a Structuring Technique: Literature Review, Modeling and Experiments," 2018, pp. 1–316, which are incorporated herein by reference in their entirety.

[0057] In another embodiment, as discussed in Choi et al., "UV Nanoimprint Lithography," Handbook of Nanofabrication, edited by Gary Wiederrecht, Elsevier Press, October 2009, see pp. 310, 149-181, a gold catalyst (e.g., catalyst 805) is used, in conjunction with an electric field, to fabricate a porous layer beneath the bathtub using an optimized MACE etchant composition after blocking all other regions except the SZ region using a polymer coating such as an inkjet and UV-curable acrylate material. Alternatively, a porous silicon layer can be fabricated in the bathtub region without an electric field using stain etching and an etchant consisting of a strong oxidizing agent such as HF and nitric acid.

[0058] In one embodiment, a porous region fabricated beneath gold can be followed by patterning and etching of the gold to create the optimal SERS pattern required for signal enhancement. An exemplary SERS pattern is discussed in Sharma et al., "SERS: Materials, Applications and the Future," Materials Today, Vol. 15, Nos. 1-2, January-February 2012, pp. 16-25. This patterning step can be performed using nanoimprint lithography and wet etching steps, as described below.

[0059] (1) After creating a porous region under the bath in the SZ portion of the wafer, the wafer is cleaned and all polymer material is removed using oxygen plasma or UV ozone cleaning.

[0060] (2) A thin (less than 10 nm) adhesive layer is coated over the entire wafer, as reported by Choi et al. in "UV Nanoimprint Lithography," Handbook of Nanofabrication, edited by Gary Wiederrecht, Elsevier Press, October 2009, pp. 310, 149-181.

[0061] (3) The imprint template containing the desired SERS pattern is imprinted onto the adhesive layer at the bottom of the “bathtub”. The template has the desired SERS pattern on the “mesa” that fits into the bathtub. Once this imprint step is complete, there is a residual polymer layer 15-40 nm thick beneath the SERS pattern, while the rest of the wafer is covered with a residual polymer film of at least 75 nm thick.

[0062] (4) Next, the residual layer (descam) etching is performed in the same manner as discussed in Figures 7 and 8A to 8D to etch the residual layer and adhesive layer, exposing the gold film in the recessed resist region.

[0063] (5) Next, the wafer is exposed to a gold wet etchant to etch the gold SERS structure at the bottom of the bath.

[0064] (6) Finally, the polymer imprint material is removed in all locations to complete the fabrication of the integrated SERS sensor on the porous silicon material in the SZ region. This allows the porous silicon to absorb solvents and buffers and sense these materials (e.g., exosomes, biomolecules, proteins, etc.).

[0065] Figure 13 is a flowchart of Method 1300 for fabricating a self-aligned pillar using the MACE process according to one embodiment of the present invention. Figures 14A to 14C show cross-sectional views for fabricating a self-aligned pillar using the MACE process using the steps described in Figure 13 according to one embodiment of the present invention.

[0066] Referring to Figures 13 in conjunction with Figures 14A to 14C, in step 1301, the MACE catalyst 1401 is deposited on the opening section of the substrate 1402, where the opening section refers to these sections on the substrate 1402 that do not include pillars 1403 (e.g., tapered pillars), as shown in Figure 14A. In one embodiment, such tapered pillars 1403 are fabricated by the MACE process for the DLD array 101. These pillars can be fabricated in a specific tapered shape using a self-aligned multi-step MACE process, as shown in Figure 14A.

[0067] In step 1302, as shown in Figure 14B, oxide 1404 is deposited and / or grown on pillar 1403, for example, along its sidewalls. In one embodiment, the sidewall oxidation step is performed using common semiconductor oxidation techniques, such as thermal oxidation or exposure to an oxygen plasma.

[0068] In step 1303, the sidewall oxide 1404 is removed (dissolved) along with a portion of the silicon 1402, as shown in Figure 14C. For example, in one embodiment, a thin wall of the formed oxide 1404 is removed using HF vapor or a short BOE immersion.

[0069] As a result of using the principles of the present invention discussed above, biomarkers and trace amounts of nanoparticles in chemical mixtures or water can be effectively detected.

[0070] The descriptions of various embodiments of the present invention have been presented for illustrative purposes only, and are not intended to be exhaustive, nor are they limited to the embodiments disclosed. Many changes and modifications will be apparent to those skilled in the art without departing from the scope and spirit of the embodiments described. The terms used herein have been selected to best describe the principles of the embodiments, their practical applications, or technical improvements to the art found in the market, or to enable those skilled in the art to understand the embodiments disclosed herein. [Explanation of Symbols]

[0071] 101 Pillar Array 102 Entrance 103 Output Stream 201A~201D Desktop Devices 202 chips 203 Chip holder 204 Sensor Zone 301A~301D area 302A~302E Output 601 Top transparent substrate 602 Microscale Gap 603 Pillar 604 Plexiglass substrate 605 Entrance hole 606 Exit hole 801 circuit board 802 Thermal Oxides 803 Resist 804 Resist Pillar 805 Catalyst 1201 Barrier layer / array 1401 MACE catalyst 1402 circuit board 1403 Pillar 1404 Oxide

Claims

1. One or more input units, into which a sample containing particles of different sizes is introduced, Multiple isolation regions, wherein the sample is pressurized as it passes through the multiple isolation regions, each of the multiple isolation regions includes a deterministic transverse displacement array, and the deterministic transverse displacement arrays in two or more of the multiple isolation regions have different etching depth profiles. Diagnostic chips, including...

2. The diagnostic chip according to claim 1, wherein the pillars in the deterministic transverse displacement array are manufactured using metal-assisted chemical etching.

3. The diagnostic chip according to claim 1, wherein the pillars in the deterministic transverse displacement array are manufactured using nanoimprint lithography.

4. The diagnostic chip according to claim 1, wherein the deterministic transverse displacement array is used for particle separation.

5. The diagnostic chip according to claim 1, wherein the pillars in the deterministic transverse displacement array taper towards the end.

6. The diagnostic chip according to claim 1, wherein the pillars in the deterministic transverse displacement array are fabricated using metal-assisted chemical etching and silicon oxidation.

7. The diagnostic chip according to claim 1, wherein the pillars in the deterministic transverse displacement array have a diameter-to-pitch ratio greater than 0.8, and the pillars are designed to prevent particle clogging in the sample.

8. Side barrier array in the deterministic transverse displacement array for particle separation The diagnostic chip according to claim 1, further comprising:

9. The diagnostic chip according to claim 1, wherein the sample comprises one of the following: blood, serum, saliva, and urine.

10. A device for separating one or more biological species, A separation region comprising a microscale or nanoscale structure, wherein the substrate beneath the separation region is nonporous, At least one output region, wherein the substrate beneath the at least one output region is porous, A device including a device.

11. Integrated surface-enhanced Raman spectroscopy (SERS) sensor with a porous silicon layer for detecting one or more biological species. The apparatus according to claim 10, further comprising:

12. The apparatus according to claim 11, wherein the porous silicon layer is designed to act as a discharge section for the sample liquid while preventing particles in the fluid from penetrating into the pores in the porous region.

13. The apparatus according to claim 10, wherein the apparatus is a deterministic transverse displacement apparatus manufactured using metal-assisted chemical etching.

14. Multiple input units, into which a sample containing particles of different sizes is introduced, and the sample includes one of the following: blood, serum, saliva, and urine. The apparatus according to claim 10, further comprising:

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