Microplates are well-known tools for chemical and biochemical analysis, wherein multiple test samples are subjected to analysis for any of a variety of applications, e.g., medical diagnostics, food, beverage and cosmetics testing, environmental monitoring, manufacturing quality control, pharmaceutical research, and basic scientific research. Microplates of this general type are often constructed as a standard 96-well assay plate, in which an eight-by-twelve array of recesses or wells is formed. The wells each constitute a cell with a generally cylindrical cell wall and a horizontal cell floor. The microplates can be formed or molded of a neutral material, e.g., glass, a polymer, or in some cases a metal or silicon. The typical ninety-six well microplate is only about three and five-eighths inch by five inches, with the wells each occupying an area of about one-twentieth of a square inches, and typically with a shallow depth, so that the required amount of the target material and any reagent would be quite limited. The microplates are of generally standard sizes to they can be used in automated robotic testing equipment. There are other standard sizes, having more or fewer wells in each microplate. Moreover, due to the need to avoid contamination, these microplates or assay plates are often considered expendible, for one-time use only.
To date, microplates or assay plates have not been provided with a microstructured metallic substrate within the wells, i.e., on the floors or walls, to render them suitable for any of the varieties of Raman scattering or Raman spectroscopy that can be used for analysis of the test sample or targets material. Suitable microplates for other types of analysis have not been optimal. In particular, a microplate readily suitable for Surface Enhanced Raman Spectroscopy or SERS has not been available, or for other forms of analysis. The problem is not limited only to Raman spectroscopy.
In our earlier co-pending U.S. patent application Ser. No. 16/156,612, now U.S. Pat. No. 10,829,846, we disclosed a process for forming a substrate suitable for SERS, e.g., forming a framework of copper oxide dendrites on a copper substrate, which are then coated or plated with a noble metal, e.g., gold, silver or equivalent. This creates metal-coated dendrites with nano-structures, favorably in a range of 50 to 200 nm. This framework of noble-metal-coated copper-oxide dendrites is well suited for SERS analysis, and for many other related analytic techniques as well. We have found that placing such a framework of noble metal microstructures on the floor and/or walls of each of the wells of the microplate array provides an excellent test bed for automated analysis of small test quantities of target materials, using SERS or other forms of analysis.
The microplate can be created by a straightforward manufacturing process, and in this case can be especially adapted for Raman spectroscopy, including but not limited to surface-enhanced Raman spectroscopy for determining the make-up of an array of various test samples. Each of the wells (i.e., cups or cells) contains a small test sample of a substance. With the microplate being dimensioned as a standard size (e.g., a regular 12×8 array of wells) the microplate permits a SERS (or other) analysis to be done automatically and in a predetermined order on each of the 96 samples to be tested. In our process, there is a layer of copper in each well, which can be deposited galvanically, electrolytically, or electrolessly, or by vapor or ion deposition. This material is subjected to a treatment, which may involve a known oxidizer, to form copper oxide, which is in the form of microscopic CuO dendrites in the range of between about 0.001 nanometer to 100 nanometers. These dendrites may be in the form of needles, fern-like structures, of fan-shaped structures, and with the dendrites present over a wide range of sizes. Then the cupric oxide dendrites are coated are coated with another metal, in this case with a noble metal or monetary metal, such as gold or silver, or another equivalent metal such as Pa, Pt, or in some cases Sn, Ni, Cu, Rh or Zn, depending on the needs of the Raman spectroscopy process and the test or target material. Thereafter the metal-coated cupric oxide dendrites are cleaned and rinsed.
The nano-structured dendrites, covered with a thin coating of noble metal upon the copper-oxide dendrites, provides a superior response for SERS analysis of the test samples.
In view of the need for new and improved ways for employing analytic technologies with high repeatability and accuracy, it is an important object of this invention to offer an economical means to create a microplate or similar arrayed test tray, coated with an appropriate microstructure of metal or metals, and/or metal oxides, to make possible an enhanced analysis that can be carried out on a routine basis.
It is another object to provide a microplate with an array of wells or test cells, in which bottoms and/or sides of the wells are given a substrate of metal and/or metal oxides, such that at the bottom and/or side of each well there exists a metal or metal oxide layer of any of the following: zinc, cadmium, nickel, lithium, silicon, gold, silver, palladium, platinum, rhodium, tin, iron, indium, copper or such other metal that may be applied as a metallic layer by electrolytic plating, electrodes plating, vacuum deposition, plasma deposition, flame spraying, immersion plating, or any other suitable method for depositing an adherent metallic layer to the bottom and/or sides of the well. The metal and/or metal oxide can exist just at the bottom or base of the well, or in some cases just at the side wall of the well. The wells are typically round, i.e., cylindrical, but can be of other geometries, as needed.
Conventionally, microplates are formed of a suitable solid material with the wells molded in. Often, the material is a molded plastic resin, such a polystyrene, but may be another suitable material such as acrylic, polypropylene, glass, silicon, or any other material compatible with the target material and the analysis to be carried out.
Most currently available microplates can be considered to be an array of mini-test tubes, simply holding the samples to be analyzed without reacting with them. In some cases, porous or semipros membranes have been added, gels with metal nano-particles have been placed in the bottoms of wells, or electrodes installed for luminance testing. In some cases, magnetic material has been included for the magnetic separation of certain biologic materials.
However, conventional microplates or variations that have been proposed cannot meet current and future technological demands. There is an unaddressed need for microplates that have surfaces that contact the test sample substances and can react with the substances in a way that aids in the analysis and assay during the testing procedure. One example of such procedure is Surface Enhanced Raman Spectroscopy, where the metallic microstructures create a plasmon that affects the spectrum for the sample and aids in identifying components in the sample. Other examples of such procedures are Tip Enhanced Raman Spectroscopy, Laser Desorption Ionization Spectrometry, X-Ray Photoelectron Spectroscopy, and X-Ray Fluorescence Microscopy. This is not an exhaustive list of the possible procedures. Particularly with Raman Spectroscopy, a modified metal surface aids in the creation of a plasmon that is essential in analysis. Certain metallic surfaces, or metaloxide surfaces, or some combinations of metals and metal oxides, are reactive with the substance of interest, or reactive with something to which the substance is attached, the reaction being physical or chemical to the subject substance of interest. The results of the physical or chemical reaction can be essential to an accurate and repeatable analysis.
A multiple-well microplate can be manufactured from a flat piece of suitable material in which the wells are molded or machined. This material may be PVC, ABS, acrylic, metal, Teflon, silicon, rubber, stainless steel, glass, glass fibers, or any other suitable material in a thickness of 0.01 inch to 1.5 inches, but preferably between about 0.10 to 0.50 inches. In this case, an array of through-holes are formed in the material, and another flat plate is attached, that plate being made of any of the above-mentioned materials. That flat plate may also be provided with metallized and treated disks at locations that are in register with the through-holes when the two plates are cemented or joined together. This forms a multiple well plate with an array of wells. These materials may be the same as is frequently used for printed circuit boards, as these are well suited to have metal and metal oxide layers deposited onto them. The microplate may have printed conductive strips connecting to some or all of the metallized disks.
In our earlier-filed U.S. patent application Ser. No. 16/156,612, filed Oct. 10, 2018, (Pub. No. US2020-0071812), we described the technology for producing a substrate that provides superior performance for use in surface-enhanced Raman spectroscopy, or SERS, by applying a suitable oxidizing agent to a copper metal substrate to produce cupric oxide dendrites, which form in a wide range of sizes extending well down into the nanoscale range, and then coating these copper oxide dendrites with another metal, preferably a monetary metal, such as gold. The resulting nano-structures of a monetary metal layer on the copper oxide nanostructures can be used to physically or chemically react with a test sample for Surface Enhanced Raman Spectroscopy. This material may also have applications with other related Raman techniques as well as in electronics, magnetics, batteries, solar cells, and others.
Many microplates of this general type have been produced, with an array of test wells (e.g., eight by twelve) or cups supported on or molded or bored into a main plate of a suitable material. In our invention the bores have the above-described nano-structure of metal-coated copper-oxide dendrides deposited onto the side walls and/or the bottoms of the wells.
A microplate, microwell plate, or multiwell is a flat plate with multiple “wells” used as small test tubes. When constructed according to the present invention, the microplate can be constructed much like a standard microplate for research and clinical diagnostic testing, and one embodiment is shown in
The surface treatment 20 within the wells 14 may be a coating achieved by electroplating, electrodes plating, immersion plating, physical vapor deposition, flame spraying, or any other suitable means to produce a suitable adherent film on the bottom and/or sides of the wells 14 of a microplate 10.
Another embodiment 110 of the microplate of this invention, as shown in
Other embodiments of this invention may also be achieved by incorporating the flat lower bottom piece 116 of plastic resin or circuit board material into the multi-well microplate during a molding process, such that the metallized disks 120 on bottom of the wells of the microplate are exposed. Bonding of the flat metal-containing piece and the side-wall piece would then be achieved.
A possible alternative construction could have the entire upper surface of the bottom piece 116 covered with the nano-structure metallic dendrites, but the described embodiment with the metallized dendrites limited to disks 120 is preferred as much less material would be needed.
A practical version of the microplate of
Another practical version of an embodiment, here of the microplate of
Many possible variations and re-configurations of microplates can incorporate the features and principles of the present invention, as defined in the appended claims.
This application claims priority under 35 U.S.C. 119(e) of Provisional Application Ser. No. 63/061392, filed Aug. 5, 2020, the disclosure of which is incorporated by reference herein. This application is also a Continuation-in-Part of co-pending U.S. patent application Ser. No. 16/156,612, filed Oct. 28, 2018 (now U.S. Pat. No. 10,829,846, Nov. 10, 2020), which claims priority under 35 U.S.C. 119(e) of Provisional Appin. Ser. No. 62/723,635, Aug. 28, 2018, the disclosure whereof is incorporated herein by reference.
Number | Date | Country | |
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63061392 | Aug 2020 | US | |
62723635 | Aug 2018 | US |
Number | Date | Country | |
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Parent | 16156612 | Oct 2018 | US |
Child | 17074793 | US |