WO2021225600A1

WO2021225600A1 - Fluid dispensing for calibrated plasmonic sensing

Info

Publication number: WO2021225600A1
Application number: PCT/US2020/032028
Authority: WO
Inventors: Fausto D'APUZZO; Raghuvir N. SENGUPTA; Steven Barcelo; Milo OVERBAY
Original assignee: Hewlett-Packard Development Company, L.P.
Priority date: 2020-05-08
Filing date: 2020-05-08
Publication date: 2021-11-11

Abstract

Systems and methods for generating test spots for plasmonic sensing are disclosed. An example apparatus includes a first fluid ejection device coupled to a first fluid vessel, and a second fluid ejection device coupled to a second fluid vessel. The apparatus also includes a processor to control the first fluid ejection device to dispense colloidal nanoparticles from the first fluid vessel onto a testing location on a substrate, and control the second fluid ejection device to dispense a target analyte from the second fluid vessel onto the testing location to form a test mixture.

Description

FLUID DISPENSING FOR CALIBRATED PLASMONIC SENSING

BACKGROUND

[0001] Plasmonic sensing is a powerful tool for trace level chemical detection. Surface-enhanced Raman spectroscopy is a plasmonic sensing technique in which Raman scattering is enhanced by a plasmonic material, such as a rough metal surface or metal nano-particles. The inclusion of the plasmonic material amplifies the Raman scattering response of the analyte, resulting in a much more sensitive test that can detect a small number of molecules or even a single molecule.

DESCRIPTION OF THE DRAWINGS

[0002] Certain exemplary embodiments are described in the following detailed description and in reference to the drawings, in which:

[0003] Fig. 1 is a schematic drawing of a system 100 for preparing test mixtures for plasmonic sensing, in accordance with an example;

[0004] Fig. 2 is another schematic drawing of a system 200 for preparing test mixtures for plasmonic sensing, in accordance with an example;

[0005] Fig. 3 is an example of a substrate with a plurality of test spots for performing plasmonic sensing;

[0006] Figs. 4A-4G depict a process for dispensing test spots for performing plasmonic sensing;

[0007] Fig. 5 is a process flow diagram summarizing a method of generating test spots for plasmonic sensing; and

[0008] Fig. 6 is a block diagram showing a medium 600 that contains logic for generating test spots for plasmonic sensing.

DETAILED DESCRIPTION

[0009] The present disclosure is related to a technique for performing plasmonic sensing such as Surface Enhanced Raman Spectroscopy (SERS). Raman spectroscopy is a technique for determining the chemical make-up of a target analyte by measuring the target analyte’s spectral response to electromagnetic radiation provided, for example, by a laser beam. In SERS, the target analyte is brought in contact with or close proximity to metal nanoparticles or nanostructures, which significantly increases the intensity of the Raman scattering and results in a much more sensitive test. Thus, plasmonic sensing techniques such as SERS are powerful tools for trace level chemical detection. However, quantification in plasmonic sensing may be difficult due to signal variability between measurements. [0010] The present disclosure describes techniques for dispensing spots of fluids on a substrate for performing plasmonic sensing. The technique involves dispensing a plurality of fluids via separate fluid ejection devices to form a mixture to be tested. The fluids can include a colloidal solution of metal nanoparticles, the target analyte, a buffer, and reference compounds. Multiple spots can be prepared on a single substrate, and multiple mixtures can be prepared by overprinting the various fluids in different proportions for different spots. This allows for the collection of multiple sets of test data, which can be processed statistically to eliminate noise and provide more accurate results. A device in accordance with the present techniques may also include an integrated spectrophotometer for performing the SERS measurements after preparation of the test spots.

[0011] The techniques described herein enable reliable and quantitative plasmonic sensing using colloidal solutions of nanoparticles. Assays using colloidal nanoparticles are typically less expensive than substrate-based techniques, but suffer even more from poor reproducibility. Generating many replicates in a single assay provides more statistically relevant results. The techniques also reduce the laborious process of manually handling combinations of different fluids like solvents, buffers, and reference solutions. In addition to generating multiple replicates, multiple combinations of target analyte, colloidal nanoparticles, and other fluids can be generated with different proportions of each. The use of fluid ejection devices (e.g., inkjet) also enables the generation of smaller test spots with smaller amounts of fluid compared to manual pipetting. Thus, the number of replicates and combinations for a single assay can be increased substantially.

[0012] Fig. 1 is a schematic drawing of a system 100 for preparing test mixtures for plasmonic sensing, in accordance with an example. The system 100 can include a plurality of dispense heads 102, each coupled to its own fluid vessel 104. The dispense heads 102 are fluid ejection devices configured to dispense fluids on a substrate 106 disposed on a stage 108. The stage 108 may be coupled to an actuator 110 that is configured to move the stage 108, enabling precise control of the position of the substrate 106 during the preparation of test spots. In some examples, the actuator 110 may be coupled to the dispense heads, and the stage can remain stationary. The substrate can be any suitable material, such as glass, plastic, paper, and others.

[0013] The dispense heads 102 may be thermal inkjet dispensers, piezoelectric inkjet dispensers, among others. Each dispense head 102 can include a microfluidic ejector 112 that dispenses a controlled volume of fluid from the corresponding vessel 104 by changing the number of droplets ejected at a particular location on the substrate 106.

[0014] Each vessel 104 can hold a different fluid or mixture of fluids to be dispensed for the preparation of test spots. Additionally, although multiple vessels 104 and dispense heads 102 are shown, the system 100 may also be configured to include a single removable dispense head 102. For example, the dispense head 102 and vessel 104 may be an integrated unit that can be inserted and removed from the system 100. In this way, the system 100 can have one dispense head at a time and the multiple fluids can be dispensed in series with different dispense heads. [0015] The fluids can include a target analyte to be tested, one or more reference solutions, colloidal nanoparticles, buffer solutions, and others. The colloidal nanoparticles may be any suitable particle known to amplify the Raman spectral response, including metal nanoparticles and others. For example, the nanoparticles may be spheroidal gold or silver nanoparticles with a diameter of 30 to 100 nanometers. Flowever, other shapes, sizes, or materials, can be used. The shape, size, and material of the nanoparticle will affect the resonant frequency of the nanoparticle. Thus, variations in these values can cause variations in the spectral response seen during testing.

[0016] The target analyte(s) is in a sample of a solution to be tested. The testing of the sample may be to determine the types of chemicals or compounds within the sample (qualitative analysis) or the amount of a chemical or compound within the sample (quantitative analysis). The reference solution is a solution prepared to have a known amount of a certain chemical or compound. The testing of the reference solution can be used to generate calibration data for determining the amount of the chemical or compound in the target analyte solution.

[0017] The buffer is a solution with salt of a weak acid or base which controls the pH, which may help to prevent or induce aggregation of the nanoparticles. For example, the buffer can be a solution of citrate, acetate or phosphate salts.

[0018] In some examples, each fluid vessel contains the same types of fluids, but mixed at different concentrations. For example, each fluid vessel may contain a mixture of the analyte, colloidal nanoparticles, a buffer, and a reference compound. However, each fluid vessel may contain a different concentration of the reference compound, for example. In this example, the test spots can be created by dispensing a certain volume of fluid from one of the vessels. In other words, in this example, each fluid vessel contains a different pre-mixed fluid mixture and each pre mixed fluid mixture is printed by itself onto one or more test spots.

[0019] In some examples, each vessel can include different types of fluids, and each test spot is created by mixing fluids from two or more fluid vessels on the substrate during printing. For example, a first vessel may contain the target analyte, the second vessel may contain a reference solution, the third vessel may contain colloidal nanoparticles, and the fourth vessel may contain the buffer solution. In this example, each test spot can be generated by printing specified volumes of the various fluids onto the same test spot. Test spots with different mixture concentrations can be created by varying the fluid volumes, firing frequencies, and drying times for each test spot or group of test spots. The concentration of the solute deposited in a single spot can be varied by altering the firing frequency of the printer so that mixtures with various concentrations of solute may be formed from a fluid with a single starting concentration.

[0020] Furthermore, although four pairs of vessels and dispense heads are shown, the system 100 may include additional pairs of vessels and dispense heads depending on the details of a specific implementation. For example, additional vessels may include different types of reference solution or different concentrations of a chemical. [0021] After the dispensed volumes are ejected onto the substrate 106, each test spot may be tested using an optical system (not shown). The optical system may be a spectrophotometer, a hyperspectral camera, a line scanning spectrophotometer, or any number of other imaging systems that can be used to obtain spectral data. The optical system may be a separate instrument or, as shown in Fig. 2, the optical system may be integrated with the system for generating the test spots.

[0022] The system 100 also includes a controller 114 configured to control ejections of droplets from the microfluidic ejector 112. The controller 114 can include a processor 116, a data store 118, and an Input/Output (I/O) system 120. The processor 116 executes instructions from the data store 118, and may be any suitable type of processor, including a microprocessor, an Application Specific Integrated circuit (ASIC), a Field Programmable Gate Array (FPGA), a Central Processing Unit (CPU), and others. In some embodiments, the data store 118 is a nonvolatile memory for both operating programs and long-term storage. In other embodiments, the data store 118 includes both volatile memory for operating programs, and a long-term data store, such as a flash memory.

[0023] The I/O system 120 is controlled by the processor 116 to perform the various system operations for dispensing test fluids on the test spots. The I/O system 120 may be used to control the actuator 110 for positioning the substrate or the dispense heads during the deposition of test spots. The I/O system 120 also couples to each of the dispense heads 102 to control the dispensing of fluid droplets onto the substrate.

[0024] The data store 118 includes modules to direct the operation of the system 100. The modules may include a dispense controller 122 that includes instructions that, when executed by the processor 116, direct the processor 116 to dispense fluids for the creation of a plurality of test spots on the substrate 106. In some examples, the dispense controller 122 may be configured to create two or more fluid mixtures by overprinting fluids from two or more of the vessels onto the same test spot. The system 100 can also include a user interface (not shown) that allows a user to create one or more fluid mixture recipes that specify the fluid types and amount of each fluid to be dispensed. The user may also be able to specify the number and arrangement of the test spots and determine the fluid mixture recipes to be dispensed for each test spot of group of test spots. The dispense controller 122 can receive the user input to determine how to generate the test spots.

[0025] It is to be understood that the block diagram of Fig. 1 is not intended to indicate that the system 100 is to include all of the components shown in Fig. 1. Rather, the system 100 can include fewer or additional components not illustrated in Fig. 1.

[0026] Fig. 2 is another schematic drawing of a system 200 for preparing test mixtures for plasmonic sensing, in accordance with an example. The system 200 is similar to the system 100 described in relation to Fig. 1 , but also includes an integrated optical system for testing the test spots and generating test results. The system 200 includes one or more dispense heads 102, each coupled to its own fluid vessel 104 and configured to dispense fluids on a substrate 106 disposed on a stage 108. The dispense heads 102 are controlled by a controller 202, which controls the fluid dispensing process as described above. Additionally, although one vessel 104 and one dispense head 102 is shown in Fig. 2, it will be appreciated that there may be multiple vessels and dispense heads as shown on Fig. 1.

[0027] In the example shown in Fig. 2, the system also includes an optical system, which includes an optical sensing device such as a spectrometer 204.

Other suitable types of optical systems include a hyperspectral camera, a line scanning spectrophotometer, and others. Further, the optical system can also include optical objectives 206 configured to collect light emitted (e.g. scattered) from the test spots on the substrate 106 and direct that light to spectrometer 204.

[0028] The spectrometer 204 is controlled by the controller 202 to test the test spots deposited on the substrate 106 after an amount of time sufficient to allow the test spots to dry. The actuator 110, in addition to positioning the substrate for fluid deposition, is also configured to translate the stage 108 to position the stage 108 adjacent to the spectrometer 204 and position each of the test spots for individual testing by the spectrometer 204.

[0029] The controller 202, in addition to the dispense control module 122, also includes an optical system control module 206 and a result calculator 208. During the testing phase, the optical system control module 208 controls the actuator 110 to positions each of the test spots for measurement. Additionally, the optical system control module 208 also controls the spectrometer 204 to perform measurements and collect test data. The test process may include individually illuminating each test spot by laser light and measuring the spectral response of the test spot. The spectral response may be an emission intensity of the light deflected by the test spot over a wavelength range of interest.

[0030] Each test spot is tested separately and the data collected and stored by the controller 202. The result calculator 210 processes the spectral data for each of the test spots and generates results. For example, the result calculator 210 can generate a calibration curve based on the spectral analysis of a reference compound within some test spots and use the calibration curve to determine the concentration of an analyte on test spots containing the analyte of interest. In some examples, the test spots include multiple replicates of a fluid mixture, and the result calculator 210 can average the results obtained from the multiple replicates. The test results can then be stored to the data store 118 and/or displayed to a user.

[0031] It is to be understood that the block diagram of Fig. 2 is not intended to indicate that the system 200 is to include all of the components shown in Fig. 2. Rather, the system 200 can include fewer or additional components not illustrated in Fig. 1.

[0032] Fig. 3 is an example of a substrate with a plurality of test spots for performing plasmonic sensing. The test spots may be deposited by the system 100 described in relation to Fig. 1 or system 200 described in relation to Fig. 2. As shown in Fig. 3, the test spots 302 have been deposited in a grid pattern over the surface of the substrate 106. In this example, the plurality of test spots include four groupings, referred to herein as group A 304, group B 306, group C 308, and group D 310. Each grouping may represent a different fluid mixture or a different amount of a fluid mixture. For example, each grouping may include a different concentration of a reference compound, a different type of reference compound, a different amount or type of colloidal nanoparticles, or a different amount of the target analyte.

[0033] The different fluid mixtures may be obtained by pre-mixing the fluid mixtures and dispensing each different fluid mixture from a different fluid dispensing device. For example, with reference to Fig. 1 , each vessel 104 may contain a different fluid mixture and each of the test spot groupings 304-310 may be dispensed from one of the dispense heads 102. In some examples, the fluid mixtures are not pre-mixed, and the different fluid mixtures used in each grouping may be obtained by over-printing fluids from different vessels 104. In this example, the different fluid mixtures for each grouping may be obtained by varying the amount of fluids dispensed from the different vessels 104.

[0034] In some examples, different groupings may be dispensed from the same fluid mixture, but may have a different volume of the fluid mixture. For example, the fluid mixture dispensed in group A 304 may be the same fluid mixture dispensed in group B 306, but group B have a higher or lower volume of dispensed fluid compared to group A.

[0035] Each grouping of test spots can also include a plurality of replicates, each replicate being a test spot intended to have the same volume of the same fluid mixture as other test spots in the same group. The testing of multiple replicates of each fluid mixture can help improve test result consistency in the presence of measurement noise.

[0036] The size, pattern, and number of test spots and groupings can be varied based on the details of a specific implementation. In some examples, the volume of fluid in each test spot may be as low as 10 picoliters (pL). However, higher fluid volumes are possible depending on the number of test spots in comparison to the surface area of the substrate. The small volume of fluid that is able to be dispensed enables a large number of test spots to be dispensed in a single assay. For example, a density of 1 to 500 spots per mm² can be dispensed. Consequently the total number of spots on a single substrate can vary between 1 and several thousands (e.g. 3000).

[0037] Figs. 4A-4G depict a process for dispensing test spots for performing plasmonic sensing. The test spots may be deposited by the system 100 described in relation to Fig. 1 or system 200 described in relation to Fig. 2. As shown in Fig. 4A, the process may begin by depositing a colloid containing plasmonic nanoparticles 400. The colloid may then be allowed to dry. The drying time will vary depending, in part, on the volume of fluid dispensed. The ability to dispense small volumes of fluid for each test spot enables the test spots to dry quickly, for example, after as little as about 1 millisecond and as slow as several seconds. [0038] After the nanoparticles colloid has dried, the process may advance to dispensing a reference compound 402 on top of the nanoparticles, as shown in Fig 4B. Fig. 4C is a top view of the substrate after the reference compound 402 has been dispensed and dried. As shown in Fig. 4C, test spots in the horizontal direction have varying volumes of the reference compound 402. This enables the collection of reference data that can be used to generate a calibration curve. Additionally, test spots in the vertical direction are replicates that have the same volume of the reference compound 402. The testing of multiple replicates allows for the collection of multiple test results for the plasmonic response of a specified fluid mixture, thus enabling the use of statistical calculations to eliminate measurement noise.

[0039] After the reference compound dries, the target analyte 404 and buffer 406 can be dispensed, as shown in Fig. 4D. The target analyte 404 and buffer 406 may be premixed prior to dispensing as a solution. The buffer solution causes the nanoparticles 400 and reference compound 402 to be re-suspended in the solution, allowing all of the fluids to mix. The re-suspension and mixing of the fluids may be assisted by the fluid burst caused by the dispensing of the buffer 406 and target analyte 404. To further assist effective re-suspension and mixing, the target analyte and buffer solution can be dispensed in series of pulses timed to promote mixing.

Fig. 4E is a top view of the substrate after the target analyte 404 and buffer solution 406 has been dispensed. For the sake of clarity, the fluids are shown as discrete masses and the buffer solution is not shown in Fig. 4E. Flowever, it will be appreciated that the dispensing process will cause some mixing of the materials. [0040] Fig. 4F shows the substrate with a plurality of test spots 408 ready to be tested. As shown in Fig. 4F, the nanoparticles, reference compounds, target analyte, and buffer have been sufficiently mixed and dried. Fig. 4G shows a top view of the substrate. At this point, the test spots 408 are ready to be tested by an optical system, such as a spectrometer. In some examples, the substrate 106 may be removed from the dispensing system (e.g., system 100) and placed in the testing area of the optical system, which may be a separate system. In some examples, the substrate 106 is moved automatically to the testing area as described in relation to the system 200 of Fig. 2. [0041] Fig. 5 is a process flow diagram summarizing a method of generating test spots for plasmonic sensing. The method may be performed the system 100 described in relation to Fig. 1 or system 200 described in relation to Fig. 2. The method may begin a block 502.

[0042] At block 502, a first fluid injection device dispenses colloidal nanoparticles on testing locations of a substrate. The testing locations may be arranged in a grid comprising several tens or even hundreds of individual test spots.

[0043] At block 504, a second fluid ejection device dispenses a target analyte on the testing location. The dispensing causes the target analyte to mix with the colloidal nanoparticles to form a test mixture.

[0044] At block 506, a detection process is performed at the testing location to determine a plasmonic response of the test mixture. Analysis of the plasmonic response may be used to determine the presence and/or concentration of a substance in the target analyte. The detection process may be performed by the same instrument or a person can manually remove the substrate from the test spot preparation device and insert the substrate in a separate detection device.

[0045] The method 500 should not be interpreted as meaning that the blocks are necessarily performed in the order shown. Furthermore, fewer or greater actions can be included in the method 500 depending on the design considerations of a particular implementation. For example, as described in greater detail above, additional fluids may be dispensed on each test spot such as buffers and reference compounds. Additionally, a plurality of replicates and additional test spots with different fluid mixture recipes may be dispensed on multiple test spots.

[0046] Fig. 6 is a block diagram showing a medium 600 that contains logic for generating test spots for plasmonic sensing. The medium 600 may be a computer- readable medium, including a non-transitory medium, that stores code that can be accessed by a processor 602 over a computer bus 604. For example, the computer- readable medium 600 can be volatile or non-volatile data storage device. The medium 600 can also be a logic unit, such as an Application Specific Integrated Circuit (ASIC), a Field Programmable Gate Array (FPGA), or an arrangement of logic gates implemented in one or more integrated circuits, for example. [0047] The medium 600 may include modules 606-612 configured to perform the techniques described herein. The dispense control module 606 can be configured to control a plurality of fluid dispensing devices for generating test spots on a substrate. The optical system control module 608 can be configured to control the operation of a plasmonic sensing device such as a spectrometer. The result calculator 610 can be configure to compute test results based on data collected by the optical system controller 608. The user interface 612 can be configured to receive user input for creating fluid mixture recipes, directing the dispensing of test spots, and displaying test results. In some embodiments, the modules 607-612 may be modules of computer code configured to direct the operations of the processor 602.

[0048] The block diagram of Fig. 6 is not intended to indicate that the medium 600 is to include all of the components shown in Fig. 6. Further, the medium 600 may include any number of additional components not shown in Fig. 6, depending on the details of the specific implementation.

[0049] While the present techniques may be susceptible to various modifications and alternative forms, the exemplary examples discussed above have been shown only by way of example. It is to be understood that the technique is not intended to be limited to the particular examples disclosed herein. Indeed, the present techniques include all alternatives, modifications, and equivalents falling within the scope of the present techniques.

Claims

CLAIMS What is claimed is:

1. An apparatus to prepare test mixtures for surface enhanced Raman spectroscopy, comprising: a first fluid ejection device coupled to a first fluid vessel; a second fluid ejection device coupled to a second fluid vessel; and a processor to: control the first fluid ejection device to dispense colloidal nanoparticles from the first fluid vessel onto a testing location on a substrate; and control the second fluid ejection device to dispense a target analyte from the second fluid vessel onto the testing location to form a test mixture.

2. The apparatus of claim 1 , wherein the test mixture is a first test mixture and the testing location is a first testing location, the processor to control the first fluid ejection device and the second fluid ejection device to generate a plurality of additional test mixtures of the target analyte and the colloidal nanoparticles at separate testing locations on the substrate.

3. The apparatus of claim 1 , comprising a third fluid ejection device coupled to a third fluid vessel, wherein the processor is to: control the first fluid ejection device to dispense colloidal nanoparticles on a second testing location of the substrate; and control the third fluid ejection device to dispense a specified amount of a reference analyte from the third fluid vessel onto the second testing location to form a reference mixture, wherein the reference analyte comprises a known concentration of a target substance.

4. The apparatus of claim 3, wherein the reference mixture is a first reference mixture, the processor further to control the first fluid ejection device and the third fluid ejection device to dispense a plurality of additional reference mixtures of the reference analyte and the colloidal nanoparticles at separate testing locations on the substrate.

5. The apparatus of claim 4, wherein the plurality of additional reference mixtures comprise different amounts of the reference analyte.

6. The apparatus of claim 1 , wherein the processor is to control a firing frequency of the first fluid ejection device and the second fluid ejection device to control a concentration of the colloidal nanoparticles and the target analyte on the substrate.

7. The apparatus of claim 1 , comprising an optical system to perform a detection process at the testing location to determine a plasmonic response of the test mixture.

8. A machine-readable storage medium encoded with instructions executable by a processor, the machine-readable storage medium comprising instructions to direct the processor to: control a first fluid ejection device to dispense colloidal nanoparticles from a first fluid vessel onto a testing location on a substrate; control a second fluid ejection device to dispense a target analyte from a second fluid vessel onto the testing location to form a test mixture; position the testing location within a test window of an optical system; receive a signal from the optical system; and process the signal to identify the target analyte based on a plasmonic response of the test mixture.

9. The machine-readable storage medium of claim 8, wherein the test mixture is a first test mixture and the testing location is a first testing location, and wherein the machine-readable storage medium comprises instructions to direct the processor to control the first fluid ejection device and the second fluid ejection device to generate a plurality of additional test mixtures of the target analyte and the colloidal nanoparticles at separate testing locations on the substrate.

10. The machine-readable storage medium of claim 8, comprising instructions to direct the processor to: control the first fluid ejection device to dispense colloidal nanoparticles on a second testing location of the substrate; and control a third fluid ejection device to dispense a specified amount of a reference analyte from a third fluid vessel onto the second testing location to form a reference mixture, wherein the reference analyte comprises a known concentration of a target substance.

11 . The machine-readable storage medium of claim 10, wherein the reference mixture is a first reference mixture, and wherein the machine-readable storage medium comprises instructions to direct the processor to control the first fluid ejection device and the third fluid ejection device to dispense a plurality of additional reference mixtures of the reference analyte and the colloidal nanoparticles at separate testing locations on the substrate, wherein the plurality of additional reference mixtures comprise different amounts of the reference analyte.

12. The machine-readable storage medium of claim 11 , comprising instructions to direct the processor to perform a quantitative analysis of the target analyte by comparing the plasmonic response of the test mixture, a plasmonic response of the first reference mixture, and a plasmonic response of each of the additional reference mixtures.

13. A method of performing surface enhanced Raman spectroscopy, comprising: dispensing, via a first fluid ejection device, colloidal nanoparticles on a testing location of a substrate; dispensing, via a second fluid ejection device, a target analyte on the testing location, wherein the dispensing causes the target analyte to mix with the colloidal nanoparticles to form a test mixture; and performing a detection process at the testing location to determine a plasmonic response of the test mixture.

14. The method of claim 13, wherein the test mixture is a first test mixture disposed on a first testing location, the method further comprising dispensing a plurality of additional test mixtures of the target analyte and the colloidal nanoparticles at separate testing locations using the first fluid ejection device and the second fluid ejection device.

15. The method of claim 13, comprising: dispensing, via the first fluid ejection device, colloidal nanoparticles on a second testing location of the substrate; dispensing, on the second testing location via a third fluid ejection device, a first amount of a reference analyte to form a reference mixture, wherein the reference analyte comprises a known concentration of a target substance; dispensing, via the first fluid ejection device, colloidal nanoparticles on a third testing location of the substrate; dispensing, on the third testing location via the third fluid ejection device, a specified second amount of the reference analyte, wherein the second amount is different from the first amount.