Normalization method

Abstract

Methods and systems for normalizing and creating correction factors for measurements in a fluorometer are described. In order to ensure that the measurement results of a plurality of channels can be properly compared, a certain calibration must be carried out. The same calibrator can run on all channels and measure fluorescence. The fluorescence of one channel can be selected as a reference. The correction factor may be calculated for each channel and used for a long period of time, even possibly for the lifetime of the instrument.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 377,167, filed on September 26, 2022. The entire contents of the above application are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to normalization methods for fluorometers. Background Art

[0004] Fluorescence is the emission of light, typically in the visible range, by a compound in response to its excitation by higher energy electromagnetic radiation. When the excited compound returns to its normal or baseline excited state, the excess energy is released as light, typically at a wavelength of less energy than the wavelength used for excitation. In applications, the emitted light signal produced during fluorescence can inform the identity and / or concentration of certain compounds within a sample. A fluorometer is an example of an analytical instrument that uses excitation and emission spectra along with intensity to analyze biological samples. Using a fluorometer, the presence and concentration of compounds such as nucleic acids and some proteins can be determined, either directly or as part of an analytical workflow for DNA, RNA, and protein. Example applications include cloning, sequencing, transfection, qPCR, and protein assays.

[0005] In a conventional fluorometer, ultraviolet excitation light is generated by an excitation light source (e.g., mercury / xenon lamp, LED, laser), which can provide an intense and continuous source of radiation, thereby allowing saturation of excitable compounds. The excitation light can be collimated to improve excitation efficiency and then directed to the biological sample of interest. Fluorescent samples or fluorescent reagents bound to non-fluorescent samples are activated by exposure to the excitation light, causing the sample to fluoresce. This fluorescent emission light is received at a photodetector, and these measurements of the amount, intensity and / or distribution of the light can be used to identify and / or estimate the concentration of the analyte in the sample.

[0006] Some fluorometers are multi-channel, meaning that multiple samples can be run at once. Each channel can have its own light source or a shared light source that is split into discrete channels, sample holders, and other components. With instruments of this nature, each channel becomes a unique measurement device in its own right, where a single sample will give different readings when measured on different channels of the same instrument. This lack of consistency between channels, if not properly addressed, can result in systematic errors between measurements. Therefore, in order to ensure that samples between different channels can be properly compared, it is necessary to calibrate each channel prior to use. This can be time consuming and use up valuable resources. Therefore, there is a need for a universal normalization for multi-channel fluorometers. Summary of the invention

[0007] An embodiment according to the present disclosure includes a method of normalizing fluorescence measurements of a multi-channel fluorometer. The method includes receiving a calibrant in a plurality of receptacles of the multi-channel fluorometer; exciting the plurality of receptacles with one or more light sources; and measuring the fluorescence level of the calibrant in each of the plurality of receptacles. The method also includes using the fluorescence level of the calibrant in a first receptacle of the plurality of receptacles to generate a correction factor for the first receptacle; using the fluorescence level from the first receptacle to create a correction factor for each of the other receptacles of the plurality of receptacles; storing the correction factors; and applying the correction factors to subsequent measurements of the fluorescence level in each respective receptacle of the plurality of receptacles.

[0008] Another embodiment includes a multi-channel fluorometer. The fluorometer includes one or more receptacles configured to receive a test solution therein; one or more light sources configured to provide excitation to the one or more receptacles; and one or more photodetectors, each photodetector configured to receive emitted light from one of the excited receptacles and measure the fluorescence level of the emitted light. The fluorometer also includes a non-transitory computer-readable storage medium coupled to the one or more receptacles, the light source, and the photodetector, the non-transitory computer-readable storage medium having stored thereon a computer program which, when executed on at least one processor, causes the at least one processor to perform a method comprising the steps of: receiving an indication that a calibrant is placed in the one or more receptacles of the multi-channel fluorometer; exciting the calibrant in the one or more receptacles with the one or more light sources; measuring the fluorescence level of each calibrant in the one or more receptacles; using the fluorescence level of the calibrant in a first receptacle of the one or more receptacles to generate a correction factor for the first receptacle; using the fluorescence level from the first receptacle to create a correction factor for each of the other receptacles of the one or more receptacles; storing the correction factors; and applying the correction factors to subsequent measurements of the fluorescence level in each corresponding receptacle of the one or more receptacles.

[0009] Yet another embodiment includes a method of normalizing fluorescence measurements. The method includes receiving a plurality of fluorescence measurements of a plurality of receptacles in a fluorometer; using the fluorescence measurements of a first receptacle of the plurality of receptacles to generate a correction factor for the first receptacle; and using the fluorescence measurements from the first receptacle to create a correction factor for each of the other receptacles of the plurality of receptacles. The method also includes storing additional correction factors; and applying the correction factors to subsequent plurality of fluorescence measurements in each respective receptacle of the plurality of receptacles.

[0010] The foregoing has outlined the features and technical advantages of the present invention in a fairly broad manner so that the following detailed description may be better understood. Additional features and advantages that form the subject matter of the claims of the present invention will be described below. It will be appreciated by those skilled in the art that the disclosed concepts and specific embodiments may be readily used as a basis for modifying or designing other structures to implement the same purposes of the present invention. It will also be appreciated by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the present disclosure as set forth in the appended claims. The novel features and other objects and advantages that are considered to be characteristic of the present invention, both in terms of its organization and method of operation, will be better understood from the following description when considered in conjunction with the accompanying drawings. However, it should be expressly understood that each of the accompanying drawings is provided for purposes of illustration and description only and is not intended to be a limitation on the scope of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more fully understand the present invention, reference is now made to the following description in conjunction with the accompanying drawings, in which:

[0012] Figure 1 A prior art fluorometer is shown;

[0013] Figure 2 A fluorometer embodiment according to the present disclosure is shown;

[0014] Figure 3 A fluorometer embodiment according to the present disclosure is shown;

[0015] 4A to 4D shows the application of correction factors to fluorescence measurements according to the present disclosure;

[0016] Figure 5 shows application of correction factors when using multiple replicate samples of a standard according to the present disclosure;

[0017] Figure 6 A flow chart showing an embodiment of a method according to the present disclosure is shown;

[0018] Figure 7 A flow chart showing an embodiment of a method according to the present disclosure is shown;

[0019] Figure 8 A flow chart showing an embodiment of a method according to the present disclosure is shown; and

[0020] Fig. 9 A flow chart of a method embodiment according to the present disclosure is shown. DETAILED DESCRIPTION

[0021] Before describing in detail various embodiments of the present disclosure, it should be understood that the present disclosure is not limited to the parameters of the systems, methods, devices, products, processes and / or kits of particular examples, which parameters can of course vary. Therefore, although certain embodiments of the present disclosure will be described in detail with reference to specific configurations, parameters, components, elements, etc., these descriptions are exemplary and should not be construed as limiting the scope of the claimed invention. In addition, the terms used herein are for the purpose of describing the embodiments and are not necessarily used to limit the scope of the claimed invention.

[0022] A fluorometer is a device capable of measuring parameters of visible spectrum fluorescence, such as the intensity and wavelength distribution of the emission spectrum resulting from spectral excitation with light. This can allow the identification and measurement of specific molecules in a medium. Figure 1 A basic fluorometer 10 is shown in FIG. The fluorometer 10 includes a light source 5 that directs light through an attenuator 15, through a primary filter 20, and onto a sample 25 in a sample holder 27. Light reflected from the sample 25 may pass through a secondary filter 30 and then be received at a detector (or photomultiplier tube) 35. The detector 35 is coupled to a reader 40 or other analysis or display component so that a user may be able to see which elements or materials are present in the sample 25 via a chart or graph. Some of the light from the light source 5 will be absorbed, while some of the light will be reflected by the sample 25. Different materials absorb and reflect different wavelengths of light. By absorbing the reflected light at the detector 35, it can be determined what materials are present in the sample 25.

[0023] Some fluorometers may include multiple samples and / or multiple light sources to perform multiple analyses simultaneously. For example, one embodiment according to the present disclosure includes an 8-channel fluorometer 100, such as Figure 2 As shown in FIG. 1 and described in detail in U.S. Patent No. 11,237,108, the fluorometer 100 can measure the fluorescence of up to eight samples simultaneously for quantification of, for example, DNA, RNA, microRNA, protein, etc.

[0024] Figure 2The fluorometer 100 includes an optical system including an excitation module 102 and an emission module 104. The excitation module 102 excites one or more samples (or fluorescent markers within the samples) to generate emission light, and the emission module 104 detects the emission light for analysis. The excitation module 102 includes: one or more excitation light sources (e.g., LED 106 and / or LED 108); a beam splitter 110 configured to direct one or more excitation light beams generated by the light source in a first direction (e.g., direction 114); a collimator or attenuator element 112; a plurality of excitation mirrors 116 configured to direct one or more excitation light beams in a second direction (e.g., direction 114B) to a plurality of excitation lenses 118; and a plurality of sample receptacles 120 configured to receive sample receptacles, the excited contents of which generate emission radiation in a third direction (e.g., direction 114C) toward a plurality of emission lenses 122, a plurality of emission filters 124, and a plurality of photodetectors 126.

[0025] The excitation module 102 can use multiple excitation light sources, such as Figure 2 A blue light emitting diode (LED) 106 and a red LED 108 are shown, but it will be understood that other types or numbers of excitation light sources including various excitation wavelengths may be used. Additional or alternative excitation light sources include, for example, lasers or mercury / xenon arc lamps. The excitation light source may be selected based on a wavelength range associated with the violet, green, yellow, or orange visible light spectrum and / or an invisible light range (such as ultraviolet light, near infrared light, or infrared light). In some embodiments, one or more excitation light sources used in the excitation module are selected based on the expected identity of the analyte to be analyzed within the biological sample.

[0026] In some embodiments, the excitation light source (e.g., LEDs 106, 108) is specifically tuned to the excitation wavelength of a predetermined fluorophore. Figure 2 In the example shown, the wavelength of the excitation light generated by the blue LED 106 and the red LED 108 is selected based on the excitation wavelength of the known fluorophore to be used in the analysis of the biological sample. Alternatively, a high-intensity light source, such as a xenon / mercury arc lamp, can be used as an excitation light source. Such lamps generate ultraviolet (UV) light and visible light, making their implementation more practical for non-specific analysis where the exact excitation wavelength or wavelength range is unknown. Light sources that produce a single excitation wavelength or a known excitation wavelength range can beneficially target known fluorophores, thereby preventing or reducing unintentional excitation of non-targeted molecules in the sample. In some embodiments, an excitation filter (e.g., a bandpass filter) can be set in front of the excitation light source as needed to narrow the wavelength range of the excitation light.

[0027] like Figure 2As shown, beam splitter 110 is used to direct two excitation light beams from LEDs 106, 108 along the same optical path (e.g., direction 114), thereby reducing the number of components and space used by excitation module 102. Alternatively, a fiber optic bundle combiner or the like can be used in place of beam splitter 110. The configuration shown can be beneficial because the size of the excitation module is reduced, so that the overall footprint of the corresponding bioanalysis device is also reduced. In addition, the ability to include one or more excitation light sources allows for increased versatility and customized configuration of the system for analyzing different samples and / or various types of fluorophores, which can correspond to different ranges of excitation light.

[0028] The excitation light is collimated by a collimator element 112 (e.g., a collimator lens or a concave / parabolic mirror) after passing through the beam splitter 110. The collimated excitation light beam is transmitted along a first direction 114 toward a plurality of excitation mirrors 116. The first direction 114 is generally parallel to the optical axis of the collimator element 112. The excitation light is reflected from the mirror 116 toward a corresponding plurality of excitation lenses 118 in the form of a plurality of individual reflected light beams. Each excitation lens 118 focuses a corresponding reflected excitation light beam, generating a focused light beam (e.g., a line focused light beam) to illuminate a sample received in a sample receptacle 120 of the emission module 104. The fluorophores within each sample are excited by the focused excitation light beam and generate emission light.

[0029] like Figure 2 As shown, a separate reflected excitation light beam is reflected from each corresponding excitation mirror and propagates in a second direction (e.g., direction 114B). In some embodiments, the second direction is not perpendicular to the first direction and forms an acute angle with the first direction, such that the reflected excitation light beam propagates in a direction returning to the collimator element 112. In contrast, some conventional fluorometer optical systems are configured to direct the light path downward, perpendicular to the light path, thereby increasing the length of the entire optical system compared to the staggered mirror configuration illustrated. Additionally, some conventional fluorometer optical systems do not include a reflective excitation mirror that allows the collimated light to continue on its initial trajectory before passing through any filters and reaching the target sample. Such embodiments result in a shorter optical system than the conventional fluorometer optical system. Figure 2 The much larger system shown. Therefore, Figure 2 The interlaced mirror configuration shown and described advantageously reduces the size of the optical system.

[0030] As mentioned above, the plurality of excitation lenses 118 generate a focused excitation beam that propagates from the excitation module 102 to the emission module 104. The emission module 104 includes a series of biological sample receptacles 120 formed as a sample block. As shown, the plurality of sample receptacles 120 are arranged in a series of evenly spaced receptacles that are aligned along an axis approximately parallel to the first direction 114 of the collimated light.

[0031] Each receptacle 120 is associated with a corresponding emission lens 122, an emission filter 124, and a photodetector 126 (e.g., a photodiode, a photomultiplier tube, a CCD / CMOS sensor, etc.) The emission module 104 is configured relative to the excitation module 102 so that each focused excitation light beam generated by the excitation module 102 propagates to a single sample receptacle disposed within the sample receptacles 120 of the emission module 104 and excites the contents thereof.

[0032] Emission light (e.g., emission radiation, fluorescent radiation) emitted by fluorescent markers or molecules within the sample contained in the receptacle 120 is collected by each of the plurality of emission lenses 122, ensuring that cross contamination of emission light from adjacent or multiple samples is prevented or minimized by focusing the emission light along the third direction 114C to the corresponding photodetector. The focused emission light then passes through a corresponding emission filter of the plurality of emission filters 124 for subsequent detection by a corresponding photodetector 126. In some embodiments, each photodetector 126 is advantageously positioned at a distance determined by the focal length of the corresponding emission lens 122 so that the emission beam passing through the emission lens is optimally focused as a line beam and reaches the target photodetector. This is beneficial in situations where one or more components are slightly misaligned by ensuring that the emission light reaches at least a portion of the surface of the photodetector lens.

[0033] As facilitated by the configuration of the optical components of the excitation module 102 and the emission module 104, the emission light is advantageously obtained in a direction different from the excitation light. It is desirable to obtain the emission light in a direction incident on the excitation light to avoid receiving direct excitation light at the emission light sensor (e.g., the photodetector 126). The emission radiation is emitted from the excited sample in all directions, and the majority of the excitation light remains directed in the second direction 114B. By placing the emission optical device in a direction transverse to (e.g., orthogonal to) the second direction 114B, a majority of the emission light can be observed in the absence of a majority of the excitation light. Any low-energy level excitation light reflected in the third direction 114C can be filtered out by the emission filter 124 (e.g., a bandpass filter) before reaching the photodetector 126.

[0034] Figure 3 Another view of a possible fluorometer embodiment is shown in . Fluorometer 300 is an 8-channel fluorometer with eight receptacles 310 for samples. Door 315 can hold the samples in place and protect them during use. Screen 330 can provide a user interface for controlling fluorometer 300 and can include a readout for viewing results.

[0035] Fluorometer 300 includes processor 340, which may be operatively coupled to screen 330, port 360, memory 350, and / or any other components or any combination thereof via bus 370. Some fluorometers may utilize Figure 3 All or a subset of the components shown in . The level of integration between components can vary. In addition, some fluorometers may include multiple instances of components, such as multiple processors, memories, transceivers, transmitters, receivers, etc.

[0036] The processor 340 is configured to process instructions and data, and may be configured to implement any sequential state machine operable to execute instructions stored as a machine-readable computer program in the memory 350. The processor 340 may be implemented as one or more hardware-implemented state machines (e.g., in discrete logic, a field programmable gate array (FPGA), an application specific integrated circuit (ASIC), etc.); programmable logic and appropriate firmware; one or more stored computer programs, general purpose processors such as a microprocessor or a digital signal processor (DSP), and appropriate software; or any combination of the above.

[0037] Screen 330 can be configured to provide or alternatively include one or more interfaces to an input device, an output device, or one or more input and / or output devices. Examples of output devices include speakers, sound cards, video cards, displays, monitors, printers, actuators, transmitters, smart cards, another output device, or any combination thereof. Examples of input devices include touch-sensitive or presence-sensing displays, cameras (e.g., digital cameras, digital video cameras, web cameras, etc.), microphones, sensors, mice, trackballs, directional keys, trackpads, rollers, smart cards, etc. Presence-sensing displays can include capacitive or resistive touch sensors to sense input from users. Sensors can be, for example, accelerometers, gyroscopes, tilt sensors, force sensors, magnetometers, optical sensors, proximity sensors, biometric sensors, etc., or any combination thereof. Output devices can use the same type of interface ports as input devices. For example, a universal serial bus (USB) port can be used to provide input devices and output devices.

[0038] Port 360 may include a power connection or a connection to other components. In some embodiments, the power source is configured as a battery or battery pack. Other types of power sources may be used, such as an external power source (e.g., an electrical outlet), a photovoltaic device, or a battery. If port 360 provides coupling to other components, port 360 may include a USB connection or similar connection. For example, a USB-C connection at port 360 may include power coupling and communication coupling to other components.

[0039] The memory 350 may include a random access memory (RAM), a read-only memory (ROM), a programmable read-only memory (PROM), an erasable programmable read-only memory (EPROM), an electrically erasable programmable read-only memory (EEPROM), a magnetic disk, an optical disk, a hard disk, a flash drive, or other types of memory. In one example, the memory 350 includes one or more application programs, such as an operating system or application, and corresponding data. The memory 350 may be configured to include multiple physical drive units, such as a redundant array of independent disks (RAID), a flash memory, a USB flash drive, an external hard drive, etc. The memory 350 may allow the fluorometer 300 to access instructions, applications, etc. stored on a transient or non-transient storage medium to unload data or upload data. The article of manufacture may be tangibly embodied on or in the memory 350, which may be or include a device-readable storage medium or a non-transient computer-readable storage medium.

[0040] Some fluorometers such as Figure 2 or Figure 3 Some fluorometers in can test multiple samples across multiple different output channels. In a multi-channel fluorometer, it may be difficult to ensure accurate results across multiple channels, multiple samples of the same material, or samples of different materials. Any difference in the function of multiple channels will distort the comparison of the results. Ensuring accurate calibration between channels and test samples can be time-consuming. For example, if a single sample is placed in multiple tubes, each tube is excited by different light / lasers, the measured output may not be equal or consistent. Standard curves can be generated using two or more standard samples of known concentrations. However, when multiple replicates of standard samples of two, three, four or more concentrations are required, such as when analyzing endotoxins, many rounds of testing will have to be performed to achieve sufficient data for calibration with a standard curve. According to embodiments of the present disclosure, methods and systems for normalizing the output of a multi-channel fluorometer are included.

[0041] FIG. 4A to FIG. 4C Helps illustrate how a fluorometer applies correction factors to measured fluorescence values ​​in a multichannel system. Figure 4AIn, chart 410 shows the result of running a set of calibrants by an 8-channel fluorometer such as QUBIT Flex fluorometer (Thermo Fisher Scientific, Waltham, MA), all of which contain the same material. The calibrants include compounds of known concentrations detectable by the fluorometer, in this example, the calibrants are 100nM ALEXA FLUOR 488 dye solutions (Thermo Fisher Scientific). Preferably, the compound is a fluorescent dye, a fluorophore, a quantum dot, a fluorescent nanoparticle, or a fluorescent gelling polymer. Exemplary fluorophores include, but are not limited to, xanthenes, fluorescein, rhodamine, rhodol, roseamine, carbopyranone, indole, indacene, borapolyazaindacene, furan, benzofuran, cyanine, benzocyanine, benzopyrilium, pyrene, coumarin, styryl, squaric acid, resorufin, anthraquinone, acridine, and benzophenoxazine. This is preferably done as the first step in setting up a new fluorometer after replacing or fixing a component, at or after factory calibration, or after a period of use to confirm or adjust calibration ratios. FIG. 4A to FIG. 4C In the example shown in , all eight wells of the QUBITFlex tube row are filled with 200 μL of the same 100 nM ALEXA FLUOR 488 dye solution, and fluorescence level measurements (expressed as RFU) are obtained for each channel. Figure 4B As can be seen from the corresponding table 420 in FIG. 4 , the uncalibrated fluorescence measurement results (RFU) of each channel CH1-CH8 are different, although they should be the same because they measure the same sample. Figure 4C , equation 430 shows how to calculate the correction factor n for each of the eight channels. 1 -n 8 In this embodiment, channel 1 (CH1) is used as the reference channel, but another channel could be used. To create each correction factor, the channel 1 fluorescence RFU (Ref1) is divided by the fluorescence RFU (RefX) of each corresponding channel. This will give a correction factor of 1 for the reference channel. In this example, n 1 = 1. For channel 2, n 2 =RFU(Ref1) / RFU(Ref2)=1.079, etc. Table 420 shows the resulting correction factors for a given data set. Figure 4A450 shows the normalized fluorescence measurement results. Because this is a calibrator, each channel should measure the same fluorescence, which is what graph 450 shows. To obtain the numbers in graph 450, the appropriate correction factor is multiplied by the measured RFU of each corresponding channel. The measured RFU in this example (normalized to channel 1) is 358 RFU (Table 420, "Corrected RFU"). Therefore, for channel 2, 358 = n 2 ×RFU(Ref2), or 358 = (1.079) × (332). The other channels can be calculated similarly. The correction factors can be stored by the fluorometer and used in subsequent experiments.

[0042] In another embodiment, the calibration factor is generated by a method that is actually opposite to the above method. In this embodiment, channel 1 is used as a reference channel again, but another channel can be used. In order to generate the correction factor, the fluorescence RFU (RefX) of the calibrant in each corresponding channel is divided by the channel 1 fluorescence RFU (Ref1). The correction factor of each channel can be stored by the fluorometer and used for subsequent experiments. In order to apply the correction factor in the sample determination, the fluorescence (RFU (Sample)) of the sample is divided by the correction factor (CF) of the channel (RefX), as shown in the following equation: RFU (Sample) / CF (RefX). Other embodiments for generating the calibration factor can utilize machine learning or can be executed on cloud-like auxiliary software. In another embodiment, the correction factor can be applied to a specific value to ensure that all instruments are normalized not only between channels, but also between instruments. This value can be the average value of the calibration factor from channel 1-8, or a predetermined value.

[0043] Once the correction factor n is calculated 1 -n 8 , they can be used for a long time, possibly even for the life of the instrument. If new components are added or damaged components are repaired, it may be necessary to run a new calibration again, depending on the type of component, such as FIG. 4A to FIG. 4C For example, a damaged and repaired lens or light source may require repeated creation of correction factors. It is possible that for some embodiments, the correction factors may be accurate for a certain number of test cycles or time period (e.g., 10,000 hours of use, or 1,000 tests, or 20 months). Once that time period / use has passed, the correction factors may need to be determined again.

[0044] To further demonstrate the utility of normalization, a serial dilution series of 100 nM ALEXA FLUOR 488 dye solution was prepared with the second blank (0 nM solution) in channel 8 (CH8) increasing in concentration from the blank in channel 1 (CH1) (i.e., 0 nM) to 100 nM in channel (CH7), and the results are shown in the graph and Figure 4D The corresponding table is shown in Figure 1. First, the diluted sample is measured only in CH1 (open circles, dashed line). This is used as a control because it does not require calibration. Next, the diluted samples are measured using CH1-CH8 without correction factors (shaded circles, thin solid line). Figure 4D As can be seen in the graph, these values ​​are not consistent with those obtained from previous measurements. Next, the fluorescence levels (RFU) from the CH1-CH8 measurements were normalized using the correction factors previously determined for this instrument (NFU, solid circles, thick solid line). Advantageously, as Figure 4D As can be seen in , these normalized values ​​overlap well with the measurements obtained from CH1 only measurements (open circles, dashed line).

[0045] Advantages of the embodiments described herein include cost and time savings. In the prior art, each channel in a multichannel fluorometer must be calibrated before each use. This consumes reagents, samples, time, and energy. According to the present disclosure, a single calibration procedure can provide correction factors that can last for months, years, or the life of the fluorometer. This consumes less material and saves time. The embodiments described can also provide the ability to generate a standard curve using multiple replicates (e.g., n≥2) of known standards of two or more concentrations. Figure 5 5 shows how multiple standards can be run simultaneously according to an embodiment of the present disclosure. According to the prior art, in order to test multiple standards 510, each standard must be run separately in a multi-channel fluorometer 580. However, according to the present disclosure, after the correction factors are determined as described, multiple standards (STD1-STD4) can be run in a single test row 550. The test row 550 may include multiple receptacles for each of STD1-STD4. Because the correction factors are known, receptacles for the same standard can be accurately compared to each other.

[0046] for Figure 5In the example shown in, when there is no proper calibration of a multichannel instrument (such as an 8-channel fluorometer), running four unique concentrations as standard (STD1-STD4) requires four groups of eight fluorescence measurements, wherein each 8-hole row is a unique concentration. For an 8-channel instrument, it is 32 samples. Advantageously, by using a correction factor as provided herein to normalize the channel, running four unique concentrations as standard can be completed with only one fluorescence measurement per concentration. For an 8-channel instrument, it is now only 8 samples. Wherein this very important determination is, for example, a regulation determination, such as endotoxin, wherein multiple replicates are required. However, this also proves to be useful for any other determination, because this will reduce the amount of the sample or reagent used and the time spent using the workflow in which the fluorescence output of each channel is separated and not normalized. For example, a standard curve using a known standard of multiple concentrations can be run simultaneously. In one example, the channels 1-8 of an 8-channel fluorometer can be used to run a single replicate of a standard 1-8, wherein the standard 1-8 is a standard of a known concentration that is incremented. In another example, channels 1-2 can be used to run two replicates of standard 1, channels 3-4 can be used to run two replicates of standard 2, channels 5-6 can be used to run two replicates of standard 3, and channels 7-8 can be used to run two replicates of standard 4. In another example using an 8-channel fluorometer, four replicates of two concentrations of standards are used, where standard 1 is measured in channels 1-4 and standard 2 is measured in channels 5-8. Another advantage of the correction factors described herein is that the two-row option (e.g., two QUBIT Flex tube rows, each containing 8 wells) allows for accommodation of three or more (e.g., N=3 or more) replicates. For example, four concentrations of standards, each with four replicates can be configured as follows: channels 1-4 of row 1 can be used for standard 1, channels 5-8 of row 1 can be used for standard 2, channels 1-4 of row 2 can be used for standard 3, and channels 5-8 of row 2 can be used for standard 4. In another example using two rows, eight standards with two replicates of each standard can be configured as follows: channels 1-2 of row 1 can be used for standard 1, channels 3-4 of row 1 can be used for standard 2, channels 5-6 of row 1 can be used for standard 3, channels 7-8 of row 1 can be used for standard 4, channels 1-2 of row 2 can be used for standard 5, channels 3-4 of row 2 can be used for standard 6, channels 5-6 of row 2 can be used for standard 7, and channels 7-8 of row 2 can be used for standard 8.

[0047] Reference has been made to an 8-channel fluorometer. However, embodiments according to the present disclosure are not limited to 8-channel embodiments. Fluorometer embodiments may include any multi-channel embodiments, such as 2-channel, 4-channel, 16-channel, 32-channel, 64-channel, or other multi-channel devices.

[0048] Figure 6 A flow chart of a possible method implementation according to the present disclosure is shown. Method 600 is a method of normalizing fluorescence measurements of a multi-channel fluorometer. Step 610 is receiving a calibrant in a plurality of receptacles of the multi-channel fluorometer. Step 620 is exciting the plurality of receptacles with one or more light sources. Step 630 is measuring the fluorescence level of each calibrant in the plurality of receptacles. Step 640 is using the fluorescence level of the calibrant in a first receptacle of the plurality of receptacles to generate a correction factor for the first receptacle. Step 650 is using the fluorescence level from the first receptacle to create a correction factor for each of the other receptacles of the plurality of receptacles. Step 660 is storing the correction factor. Step 670 is applying the correction factor to subsequent measurements of the fluorescence level in each respective receptacle of the plurality of receptacles.

[0049] Figure 7 A flow chart of another possible method embodiment according to the present disclosure is shown. Method 700 is a method of normalizing fluorescence measurements. Step 710 is receiving a plurality of fluorescence measurements of a plurality of receptacles in a fluorometer. Step 720 is using the fluorescence measurement of a first receptacle in the plurality of receptacles to generate a correction factor for the first receptacle. Step 730 is using the fluorescence measurement from the first receptacle to create a correction factor for each of the other receptacles in the plurality of receptacles. Step 740 is storing the correction factor. Step 750 is applying the correction factor to a subsequent plurality of fluorescence measurements in each respective receptacle in the plurality of receptacles.

[0050] Figure 8A method for creating a standard curve using a normalized correction factor and a series of standard solutions of known concentrations is shown, which can be used to determine the concentration of an analyte of interest. Method 800 includes step 810, which is to receive a plurality of first fluorescence measurements of a plurality of receptacles in a fluorometer, each receptacle containing a calibrant. Step 820 is to create a correction factor for the first receptacle using the fluorescence measurement of the first receptacle in the plurality of receptacles. Step 830 is to create a correction factor for each receptacle in the other receptacles using the fluorescence measurement from the first receptacle. Step 840 is to store the correction factor. Step 850 of method 800 is to receive a plurality of second fluorescence measurements of a plurality of receptacles, each receptacle containing a standard solution of known dilution or concentration. Step 860 is to apply the correction factor to a plurality of second fluorescence measurements of those known standard solutions. Step 870 is to plot a standard curve using the normalized data points of the standard solutions to create a curve of concentration versus fluorescence. Then, step 880 is to measure the fluorescence of a sample containing the analyte of interest. Then, step 890 is to normalize the measurement results using the correction factor, and step 895 is to plot the measurement results relative to the standard curve to determine the concentration of the analyte of interest. The analyte of interest can be a biomolecule. In certain embodiments, the biomolecule is selected from a protein, a peptide, an amino acid, an enzyme, a toxin, a lectin, a lipopolysaccharide, a nanoparticle, a virus, an extracellular vesicle, a nucleic acid, a polynucleotide, an oligonucleotide, a single-stranded DNA, a double-stranded DNA, an RNA, or a microRNA.

[0051] As mentioned above, the creation of a standard curve is preferably completed using an array of standard solutions of known concentration. The array can be composed of a single instance of a standard solution of each concentration or two or more replicates of each concentration. In certain embodiments, these standard arrays are prepackaged and placed in a row of fluorometer tubes connected with a visible orientation to ensure that they are properly placed in the device. This type of standard array eliminates the need for transferring standards from a single vial of a solution, and prevents the standard from being wrongly placed in the test device.

[0052] Fig. 9Another method embodiment 900 according to the present disclosure is shown. Method 900 includes a method for testing fluorescence according to multiple standards in a single test. Step 910 is to receive multiple first fluorescence measurements of multiple receptacles in a fluorometer, each receptacle containing a calibrant. Step 920 is to use the fluorescence measurement of the first receptacle in the multiple receptacles to generate a correction factor for the first receptacle. Step 930 is to use the fluorescence measurement from the first receptacle to create a correction factor for each of the other receptacles in the multiple receptacles. Step 940 is to store the correction factor. Step 950 is to receive multiple second fluorescence measurements of multiple receptacles, wherein one or more first receptacles in the multiple receptacles contain a first standard of each concentration, and one or more second receptacles in the multiple receptacles contain a second standard of each concentration. Step 960 is to apply the correction factor to multiple second fluorescence measurements. In one possible embodiment of method 900, there are eight receptacles. And in one possible variation, two receptacles are used for each of four different standards. Other variations are possible.

[0053] Computer system of the present disclosure

[0054] It should be understood that computer systems increasingly take a variety of forms. In this specification and in the claims, the term "controller", "computer system" or "computing system" is broadly defined to include any device or system or combination thereof, which includes at least one physical and tangible processor and a physical and tangible memory capable of having computer executable instructions that can be executed by the processor. As an example and not a limitation, the term "computer system" or "computing system" as used herein is intended to include personal computers, desktop computers, laptop computers, tablet computers, handheld devices (e.g., mobile phones, PDAs, pagers), microprocessor-based or programmable consumer electronics, microcomputers, mainframe computers, multiprocessor systems, network personal computers, distributed computing systems, data centers, message processors, routers, switches, and even devices that have not traditionally been considered computing systems, such as wearables (e.g., glasses) or cloud-based applications.

[0055] The memory may take any form and may depend on the nature and form of the computing system. The memory may be physical system memory, including volatile memory, non-volatile memory, or some combination of the two. The term "memory" may also be used herein to refer to non-volatile mass storage devices, such as physical storage media.

[0056] There are also a number of structures on a computing system that are often referred to as "executable components." For example, a computing system's memory may include executable components. The term "executable component" is a name for a structure that is well understood by those of ordinary skill in the computing arts to be software, hardware, or a combination thereof.

[0057] For example, when implemented in software, it will be understood by those skilled in the art that the structure of an executable component may include software objects, routines, methods, etc. that can be executed by one or more processors on a computing system, whether such executable components exist in the memory heap of the computing system, or whether the executable components exist on a computer-readable storage medium. The structure of the executable component exists on a computer-readable medium in such a form that when it is executed by one or more processors of the computing system, it can be operated to cause the computing system to perform one or more functions, such as the functions and methods described herein. Such a structure can be directly computer-readable by the processor - just as the case where the executable component is binary. Alternatively, the structure can be constructed to be interpretable and / or compiled (whether in a single level or in multiple levels) to generate such a binary that is directly interpretable by the processor.

[0058] The term "executable component" is also well understood by those of ordinary skill to include structures that are implemented in hardware logic components in a dedicated or near-dedicated manner, such as in a field-programmable gate array (FPGA), an application-specific integrated circuit (ASIC), a program-specific standard product (ASSP), a system on a chip (SoC), a complex programmable logic device (CPLD), or any other dedicated circuit. Therefore, the term "executable component" is a term for structures that are well understood by those of ordinary skill in the computing arts, whether implemented in software, hardware, or a combination thereof.

[0059] The terms "component", "service", "engine", "module", "control", "generator", etc. may also be used in this specification. As used in this specification and in this context, these terms (whether expressed with or without modifying clauses) are also intended to be synonymous with the term "executable component" and therefore also have structures that are well understood by those of ordinary skill in the computing arts.

[0060] Although not all computing systems require a user interface, in some embodiments, a computing system includes a user interface for conveying information from a user / convey information to a user. The user interface may include an output mechanism and an input mechanism. The principles described herein are not limited to accurate output mechanisms or input mechanisms, because this will depend on the nature of the device. However, the output mechanism may include, for example, a speaker, a display, a tactile output, a projection, a hologram, etc. The example of an input mechanism may include, for example, a microphone, a touch screen, a projection, a hologram, a camera, a keyboard, a stylus, a mouse or other indicator input, any type of sensor, etc.

[0061] Thus, the embodiments described herein may include or utilize a dedicated or general purpose computing system. The embodiments described herein also include physical and other computer readable media for carrying or storing computer executable instructions and / or data structures. Such computer readable media may be any available media that can be accessed by a general purpose or special purpose computing system. A computer readable medium that stores computer executable instructions is a physical storage medium. A computer readable medium that carries computer executable instructions is a transmission medium. Thus, by way of example and not limitation, the embodiments disclosed or contemplated herein may include at least two distinct types of computer readable media: a storage medium and a transmission medium.

[0062] Computer-readable storage media include RAM, ROM, EEPROM, solid-state drives ("SSD"), flash memory, phase-change memory ("PCM"), CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other tangible physical storage media that can be used to store desired program code in the form of computer-executable instructions or data structures and that can be accessed and executed by a general or special-purpose computing system to implement the functions disclosed in the present invention. For example, computer-executable instructions may be embodied on one or more computer-readable storage media to form a computer program product.

[0063] Transmission media may include networks and / or data links that can be used to carry the desired program code in the form of computer-executable instructions or data structures, and can be accessed and executed by general or special computing systems. The above combinations should also be included in the scope of computer-readable media.

[0064] Additionally, program code in the form of computer-executable instructions or data structures may be automatically transferred from transmission media to storage media (or vice versa) upon reaching various computing system components. For example, computer-executable instructions or data structures received over a network or data link may be buffered in RAM within a network interface module (e.g., a "NIC") and then ultimately transferred to computing system RAM and / or less volatile storage media at the computing system. Thus, it should be understood that storage media may be included in computing system components that also utilize, or even primarily utilize, transmission media.

[0065] It will also be appreciated by those skilled in the art that a computing system may also contain a communication channel that enables the computing system to communicate with other computing systems, for example, over a network. Thus, the methods described herein may be practiced in a network computing environment having many types of computing systems and computing system configurations. The disclosed methods may also be practiced in a distributed system environment, where local and / or remote computing systems linked over a network (via a hardwired data link, a wireless data link, or a combination of a hardwired data link and a wireless data link) all perform tasks. In a distributed system environment, processing, memory, and / or storage capabilities may also be distributed.

[0066] It will also be appreciated by those skilled in the art that the disclosed methods may be practiced in a cloud computing environment. A cloud computing environment may be distributed, but this is not required. When distributed, a cloud computing environment may be distributed internationally within an organization and / or have components owned across multiple organizations. In this specification and the appended claims, "cloud computing" is defined as a model for enabling on-demand network access to a shared pool of configurable computing resources (e.g., networks, servers, storage devices, applications, and services). The definition of "cloud computing" is not limited to any of the other numerous advantages that may be obtained from such a model when properly deployed.

[0067] The cloud computing model can consist of various characteristics, such as on-demand self-service, broad network access, resource pooling, rapid elasticity, measured services, etc. The cloud computing model can also appear in the form of various service models, such as software as a service ("SaaS"), platform as a service ("PaaS"), and infrastructure as a service ("IaaS"). The cloud computing model can also be deployed using different deployment models, such as private cloud, community cloud, public cloud, hybrid cloud, etc.

[0068] List of abbreviations for defined terms

[0069] To aid understanding of the scope and content of this written specification and the appended claims, selected terms are defined directly below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0070] As used herein, the terms "approximately," "about," and "substantially" refer to quantities or conditions that are close to the specifically stated quantity or condition that still performs a desired function or achieves a desired result. For example, the terms "approximately," "about," and "substantially" may refer to quantities or conditions that are less than 10%, or less than 5%, or less than 1%, or less than 0.1%, or less than 0.01% of the deviation from the specified quantity or condition.

[0071] Various aspects of the present disclosure, including devices, systems, and methods, may be described with reference to one or more embodiments or implementations that are exemplary in nature. As used herein, the term "exemplary" means "serving as an example, instance, or illustration," and should not necessarily be interpreted as being preferred or advantageous over other embodiments disclosed herein. In addition, references to "an embodiment" of the present disclosure or the invention include specific references to one or more embodiments thereof, and vice versa, and such references are intended to provide illustrative examples rather than to limit the scope of the invention, which is indicated by the appended claims rather than the following description.

[0072] As used in this specification, words appearing in the singular encompass their plural counterparts, and words appearing in the plural encompass their singular counterparts, unless implicitly or explicitly understood or stated otherwise. Therefore, it should be noted that, unless the context otherwise clearly stipulates, as used in this specification and the appended claims, the singular forms "one", "one" and "said" include plural indicators. For example, a reference to a singular referent (e.g., "widget") includes one, two or more referents, unless otherwise implicitly or explicitly understood or stated. Similarly, unless the directory and / or context clearly stipulate otherwise, a reference to multiple referents should be interpreted as including a single referent and / or multiple referents. For example, a reference to a plural referent (e.g., "widget") does not necessarily require multiple such referents. On the contrary, it should be understood that, unless otherwise stated, one or more referents are contemplated herein, independent of the number of inferred referents.

[0073] As used herein, directional terms such as "top", "bottom", "left", "right", "up", "down", "upper", "lower", "proximal", "distal", "adjacent", etc. are used herein only to indicate relative directions and are not intended to otherwise limit the scope of the present disclosure and / or the claimed invention.

[0074] in conclusion

[0075] It should be understood that, unless otherwise implicitly or explicitly understood or stated, for any given component or embodiment described herein, any possible candidates or alternatives listed for that component may generally be used individually or in combination with each other. In addition, it should be understood that, unless otherwise implicitly or explicitly understood or stated, any listing of such candidates or alternatives is merely exemplary and non-restrictive.

[0076] In addition, unless otherwise indicated, the numerals used in the specification and claims for expressing quantity, composition, distance or other measured values ​​should be understood to be modified by the term "about", as the term is defined in this article. Therefore, unless otherwise indicated, the numerical parameters set forth in this specification and the appended claims are approximate values ​​that can be varied with the desired properties that are attempted to be obtained by the subject matter proposed herein. At the lowest level, and without attempting to limit the scope of the application of the principle of equivalents to the claims, each numerical parameter should at least be interpreted according to the number of reported significant figures and by applying ordinary rounding techniques. Although the numerical range and parameters of the wide range of the subject matter proposed herein are set forth are approximate values, the numerical values ​​set forth in the specific examples are reported as accurately as possible. However, any numerical value inherently contains some errors that are necessarily caused by the standard deviations present in its corresponding test measurements.

[0077] Any headings and sub-headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description or the claims.

[0078] The terms and expressions that have been adopted herein are used as descriptive terms rather than limiting, and when such terms and expressions are used, it is not intended to exclude any equivalents of the features shown and described or parts thereof, but it should be recognized that various modifications are possible within the scope of the invention as listed. Therefore, it should be understood that although the present invention has been particularly disclosed through preferred embodiments, exemplary embodiments and optional features, those skilled in the art may make modifications and changes to the concepts disclosed herein, and such modifications and changes are considered to be within the scope of the invention as defined by the attached items. The specific embodiments provided herein are examples of useful embodiments of the present invention, and various changes and / or modifications of the inventive features described herein and additional applications of the principles described herein that will occur to those skilled in the relevant art and possess the present disclosure may be made to the illustrated embodiments without departing from the spirit and scope of the present invention as defined by the items, and these are considered to be within the scope of the present disclosure.

[0079] It should also be understood that the system, device, product, kit, method and / or process according to certain embodiments of the present disclosure may include, be incorporated into or otherwise include the properties or features (e.g., components, members, elements, parts and / or parts) described in other embodiments disclosed and / or described herein. Therefore, the various features of certain embodiments may be compatible, combined, incorporated into and / or incorporated into other embodiments of the present disclosure with other embodiments of the present disclosure. Therefore, certain features are disclosed with reference to the specific embodiments of the present disclosure, which should not be considered as limiting the application of the features to or inclusion in the specific embodiments. On the contrary, it should be understood that other embodiments may also include the features, members, elements, parts and / or parts without departing from the scope of the present disclosure.

[0080] In addition, unless a feature is described as being required to be combined with another feature, any feature herein may be combined with any other feature of the same or different embodiments disclosed herein. In addition, various well-known aspects of illustrative systems, methods, devices, etc. are not described in particular detail herein to avoid obscuring various aspects of the example embodiments. However, such aspects are also contemplated herein.

[0081] All references cited in this application are hereby incorporated by reference in their entirety to the extent that they are not inconsistent with the disclosure in this application. It will be apparent to those of ordinary skill in the art that methods, devices, device elements, materials, procedures and techniques other than those specifically described herein can be applied to the practice of the present invention as widely disclosed herein without the need for excessive experimentation. All functional equivalents known in the art of the methods, devices, device elements, materials, procedures and techniques specifically described herein are intended to be encompassed by the present invention.

[0082] When a group of materials, compositions, components or compounds is disclosed herein, it is understood that all individual members of those groups and all subgroups thereof are disclosed separately. When Markush groups or other groupings are used herein, all individual members of the group and all possible combinations and subcombinations of the group are intended to be individually included in the disclosure. Unless otherwise indicated, each formulation or combination of components described or demonstrated herein can be used to practice the present invention. Whenever a range such as a temperature range, a time range or a composition range is given in the specification, all intermediate ranges and subranges and all individual values ​​included in the given range are intended to be included in the disclosure. All changes within the meaning and equivalent range of the item will be included in its scope.

[0083] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and modifications may be made without departing from the spirit and scope of the present invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, material compositions, modes, methods and steps described in the specification. It will be readily understood by those of ordinary skill in the art from the disclosure of the present invention that processes, machines, manufactures, material compositions, modes, methods or steps that currently exist or will be developed later to perform functions substantially the same as the corresponding embodiments described herein or to achieve substantially the same results may be utilized according to the present invention. Therefore, the appended claims are intended to include such processes, machines, manufactures, material compositions, modes, methods or steps within their scope.

Claims

1. A method for normalizing fluorescence measurement results of a multi-channel fluorometer, the method comprising: receiving calibrants in a plurality of receptacles of the multi-channel fluorometer; energizing the plurality of receptacles with one or more light sources; measuring a fluorescence level of each calibrant in the plurality of receptacles; generating a correction factor for a first receptacle of the plurality of receptacles using the fluorescence level of the calibrant in the first receptacle; using the fluorescence level from the first receptacle to create a correction factor for each of the other receptacles of the plurality of receptacles; storing the correction factor; and The correction factor is applied to subsequent measurements of the fluorescence level in each respective receptacle of the plurality of receptacles. 2 . The method of claim 1 , wherein creating a correction factor comprises dividing the fluorescence level of the first receptacle by the fluorescence level of each respective receptacle of the plurality of receptacles.

3. The method of claim 1, further comprising generating a standard curve based at least in part on the correction factor.

4. The method of claim 3, wherein generating the standard curve comprises applying the correction factor obtained for each of the plurality of receptacles to subsequent measurements of fluorescence levels of standard solutions of known concentration in each respective receptacle of the plurality of receptacles.

5. The method of claim 3, wherein generating the standard curve comprises using two or more replicate samples of each concentration of the standard solution. The method of claim 4 , wherein the standard solution comprises biomolecules.

7. The method of claim 6, wherein the biomolecule is selected from a protein, a peptide, an amino acid, an enzyme, a toxin, a lectin, a lipopolysaccharide, a nanoparticle, a virus, an extracellular vesicle, a nucleic acid, a polynucleotide, an oligonucleotide, a single-stranded DNA, a double-stranded DNA, an RNA or a microRNA.

8. The method according to claim 1, further comprising: Subsequent measurements of the fluorescence level of a sample containing an analyte of interest are obtained in one or more of the plurality of receptacles, and the correction factors obtained for the one or more of the plurality of receptacles are applied.

9. The method of claim 8, wherein the analyte of interest is a biomolecule.

10. The method of claim 9, wherein the biomolecule is selected from a protein, a peptide, an amino acid, an enzyme, a toxin, a lectin, a lipopolysaccharide, a nanoparticle, a virus, an extracellular vesicle, a nucleic acid, a polynucleotide, an oligonucleotide, a single-stranded DNA, a double-stranded DNA, an RNA or a microRNA.

11. The method of claim 1 , wherein the calibration object is selected based on one or more types of light comprising the one or more light sources.

12. The method of claim 11, wherein the calibrant is selected such that it will fluoresce when excited by the one or more types of light.

13. The method of claim 1, wherein the calibrant comprises a fluorescent dye, a fluorophore, a quantum dot, a fluorescent nanoparticle, or a fluorescent gelling polymer.

14. A multi-channel fluorometer, comprising: one or more receptacles configured to receive a test solution therein; one or more light sources configured to provide excitation to the one or more receptacles; one or more photodetectors, each photodetector configured to receive emission light from one of the excited receptacles and measure a fluorescence level of the emission light; as well as A non-transitory computer-readable storage medium coupled to the one or more receptacles, the light source, and the light detector, the non-transitory computer-readable storage medium having a computer program stored thereon, the computer program, when executed on at least one processor, causing the at least one processor to perform a method comprising the steps of: receiving an indication that a calibrant is placed in the one or more receptacles of the multi-channel fluorometer; exciting the calibrant in the one or more receptacles with the one or more light sources; measuring the fluorescence level of each calibrant in the one or more receptacles; generating a correction factor for a first receptacle of the one or more receptacles using the fluorescence level of the calibrant in the first receptacle; using the fluorescence level from the first receptacle to create a correction factor for each of the other receptacles of the one or more receptacles; storing the correction factor; and The correction factor is applied to subsequent measurements of the fluorescence level in each respective one of the one or more receptacles.

15. The multi-channel fluorometer of claim 14, wherein creating a correction factor comprises dividing the fluorescence level of the first receptacle by the fluorescence level of each of the other receptacles of the one or more receptacles.

16. The multi-channel fluorometer of claim 14, wherein the one or more receptacles include eight receptacles.

17. The multi-channel fluorometer of claim 14, wherein the method further comprises storing the first and additional conversion factors until one of the one or more light sources is replaced.

18. The multi-channel fluorometer of claim 14, wherein the method further comprises storing the first and additional conversion factors until the multi-channel fluorometer is powered off.

19. The multi-channel fluorometer of claim 14, wherein the method further comprises storing the first and additional conversion factors until the multi-channel fluorometer has been in operation for a predetermined length of time.

20. The multi-channel fluorometer of claim 19, wherein the predetermined length of operating time is 10,000 hours.

21. The multi-channel fluorometer of claim 14, wherein the method further comprises displaying the measurement of fluorescence level on a user interface.

22. The multi-channel fluorometer of claim 14, wherein the method further comprises displaying a graph of the measurements of fluorescence levels on a user interface.

23. The multi-channel fluorometer of claim 14, wherein the subsequent measurements include measurements of one or more replicate samples of a standard solution of known concentration.

24. The multi-channel fluorometer of claim 22, wherein the standard solution comprises biomolecules.

25. The multi-channel fluorometer of claim 23, wherein the biomolecule is selected from proteins, peptides, amino acids, enzymes, toxins, lectins, lipopolysaccharides, nanoparticles, viruses, extracellular vesicles, nucleic acids, polynucleotides, oligonucleotides, single-stranded DNA, double-stranded DNA, RNA, or microRNA.

26. The multi-channel fluorometer of claim 14, wherein the subsequent measurements include measurements of a sample containing an analyte of interest.

27. The multi-channel fluorometer of claim 26, wherein the analytes of interest comprise biomolecules.

28. The multi-channel fluorometer of claim 27, wherein the biomolecule is selected from proteins, peptides, amino acids, enzymes, toxins, lectins, lipopolysaccharides, nanoparticles, viruses, extracellular vesicles, nucleic acids, polynucleotides, oligonucleotides, single-stranded DNA, double-stranded DNA, RNA, or microRNA.

29. A method for normalizing fluorescence measurement results, the method comprising: receiving a plurality of fluorescence measurements of a plurality of receptacles in a fluorometer; using the fluorescence measurement of a first receptacle of the plurality of receptacles to generate a correction factor for the first receptacle; using the fluorescence measurement from the first receptacle to create a correction factor for each of the other receptacles of the plurality of receptacles; storing the correction factor; and The correction factor is applied to a subsequent plurality of fluorescence measurements in each respective receptacle of the plurality of receptacles.

30. The method of claim 29, wherein creating a correction factor comprises dividing the fluorescence measurement of the first receptacle by the fluorescence measurement of each respective receptacle of the plurality of receptacles.

31. The method of claim 30, further comprising calculating a standard curve for the subsequent plurality of fluorescence measurements.

32. The method of claim 30, further comprising sending the corrected subsequent plurality of fluorescence measurements to a graphical interface.

33. A method for testing fluorescence against multiple standards in a single test, the method comprising: receiving a plurality of first fluorescence measurements of a plurality of receptacles in a multi-channel fluorometer, each receptacle containing a calibrant; using the fluorescence measurement of a first receptacle of the plurality of receptacles to generate a correction factor for the first receptacle; using the fluorescence measurement from the first receptacle to create a correction factor for each of the other receptacles of the plurality of receptacles; storing the correction factor; receiving a plurality of second fluorescence measurements of the plurality of receptacles, wherein one or more first receptacles of the plurality of receptacles contain respective concentrations of a first standard and one or more second receptacles of the plurality of receptacles contain respective concentrations of a second standard; and The correction factor is applied to the plurality of second fluorescence measurements.

34. The method of claim 33, wherein the plurality of second fluorescence measurements further comprises one or more third receptacles of the plurality of receptacles comprising respective concentrations of third standards and one or more fourth receptacles of the plurality of receptacles comprising respective concentrations of fourth standards.

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