Quality verification of well plate background fluorescence for a single molecule detection instrument

Abstract

A Method and a system to measure and verify the background fluorescence of well plates for use with a high-sensitivity optical spectrometry instrument via a computer, comprising of the following steps of Inserting the well plate into an apparatus which is connected to the computer and applies the scan method and data collection of the high-sensitivity optical spectrometry instrument; Forwarding the collected data to the computer where a software analyzes the data to determine the background fluorescence of the scanned well plate and checks if the background fluorescence exceeds a given maximum tolerance level; and Sorting out the well plate if the given maximum tolerance level has been exceeded.

Description

Quality verification of well plate background fluorescence for a single molecule detection instrumentBACKGROUND OF THE INVENTION

[0001] The disclosed invention relates to a method and a system for quantifying autofluorescent signal generated by well plate.

[0002] The invention belongs to the technical field of Protein Detection.

[0003] Sensitive optical instrumentation, such as fluorescence microscopes, single molecule detection instruments, or Raman spectrometers can be significantly influenced by autofluorescence, or ‘background’ from the well plate used for sample holding. Variation in background will impact fluorescent signal from target fluorescent labels, thus affecting accuracy of interpolated measurements and precision between sample replicates. For high sensitivity instruments, there is a need for uniform, low magnitude background in well plates to provide consistent measurement results.

[0004] One example of such a high sensitivity instruments is known from the yet unpublished US Provisional application 63 / 605,656 which discloses a Method and a detection system for detecting protein concentrations in a prepared assay solution consisting of several components, arranged after each after, the components being a fiber coupled laser for emitting laser light, a laser tube lens to focus the laser light, a laser cleanup filter for blocking non-laser wavelengths, a first dichroic filter for passing the laser light and reflecting fluorescence light, an objective lens to focus the laser light to a small excitation volume, the objective also collecting fluorescence light emitted from a sample, a pre-confocal and a post-confocal lens with a pinhole positioned between them to focus and re-focus the fluorescent light, a second dichroic filter, a focus photodiode for auto-focusing on the sample, at least one bandpass filter for spectrum filtering of the fluorescent light, at least one fiber focus lensfor coupling the fluorescent light to an optical fiber, at least one fiber patch cables to couple fluorescently focused light to atleast one computer-based detector for signal detection, wherein the sample is located after the objective lens, the second dichroic filter, the focus photodiode and the at least one bandpass filter are forming a De-Multiplexer for the incoming fluorescence light and the at least one bandpass filter, at least one fiber focus lens, at least one fiber patch cables and at least one computer-based detector always consist of same the number and one of each of them are located along a respective fluorescence light.

[0005] There are numerous examples of commercial well plate manufacturers who market ‘low background’ or ‘low autofluorescence’ substrate materials.These marketing claims are seldom verified in a quantifiable manner, and often relative plots of background intensity of one substrate material to another are shared. In many cases the manufacturer may only perform limited and low sensitivity testing of the substrate background, perhaps in a first article inspection, and as such variations in the background are susceptible to shift over time.

[0006] Even ideal substrate materials, such as cyclo-olefins, can show background variations over time, likely due to variations in production. Additional variations from manufacture of the well plate can also occur; potential sources include heat / pressure related processing effects and contamination from metal / plastic debris and oils, adhesives, or release agents.

[0007] There is no known process for strict measurement and verification of well plate background prior to use on an instrument platform. The task of this invention is therefore to disclose a method to continuously verify background fluorescence of well plates to ensure high quality results of their use in a sensitive instrumentation platform.BRIEF SUMMARY OF THE INVENTION

[0008] This task can be solved by a Method to measure and verify the background fluorescence of well plates for use with a high-sensitivity optical spectrometry instrument via a computer, comprising the following steps ofInserting the well plate into an apparatus which is connected to the computer and applies the scan method and data collection of the high-sensitivity optical spectrometry instrument; Forwarding the collected data to the computer where a software analyzes the data to determine the background fluorescence of the scanned well plate and checks if the background fluorescence exceeds a given maximum tolerance level; and Sorting out the well plate if the given maximum tolerance level has been exceeded. Implementation of this process for a high sensitivity optical spectrometry instrument has enabled a quantification of the effects of background on instrument measurement sensitivity. Experimental studies and Monte Carlo simulations show that even high-quality well plates may have background variations which increase signal CV’s greater than 20%, thus severely affecting signal accuracy and precision. This invention therefore introduces a computer-controlled methodology for assessing the background fluorescence in well plates, which involves an interface between a scanning apparatus and a computer. The method is characterized by its step-by-step process: inserting the well plate into the scanning apparatus, scanning the plate to collect fluorescence data, and then utilizing software to analyze this data. The software's role is to evaluate whether the background fluorescence is within acceptable limits, and to identify and reject any well plates that exceed these predetermined tolerance levels. The primary feature of this invention is the integration of hardware and software to automate the quality control process, ensuring that only well plates with acceptable background fluorescence are used, which is critical for sensitive analytical measurements. Due to the effort necessary to perform this method, it is advised to use it only at random on a small percentage of the available well plates. That should not result in a higher risk, since usually the well plates from the same load or charge are very similar in their properties.

[0009] Advantageous and therefore preferred further developments of this invention emerge from the associated sub claims and from the description and the associated drawings.

[0010] One of those preferred further developments of the disclosed method comprise that the application of the scan method and data collection is performed either at a manufacturer, inventory, or QC site prior to the delivery of the well plate to a customer. This delineates the adaptability of the method previously described. The additional feature includes the method's application across different stages of the well plate's lifecycle - at the manufacturer, in inventory stages, or at quality control sites. This provides flexibility in the quality assurance process, enabling early detection and rejection of substandard well plates before they reach the customer, thus safeguarding the integrity of subsequent scientific analyses.

[0011] Another one of those preferred further developments of the disclosed method comprise that the application of the scan method and data collection is performed at the customer location in the customer instrument in an automated form. This enables the same verification process to be executed at the customer's site with their own instrumentation. The distinguishing feature here is the automation of the verification process on-site, which eliminates the dependency on external quality control measures and provides immediate validation of well plate quality, leading to enhanced workflow efficiency.

[0012] Another one of those preferred further developments of the disclosed method comprise that as apparatus a Custom HW is used which is designed or modified from an existing platform. This feature specifies the use of a custom- designed hardware apparatus for the method. It centers about the customization of the hardware, which may be either a bespoke design or a modification of an existing system, tailored to enhance the control and precision of the scanning process for different types of epi-fluorescence instruments and well plates.

[0013] Another one of those preferred further developments of the disclosed method comprise that as apparatus a modified high-sensitivity optical spectrometry instrument is used. This features the modification of existing high- sensitivity optical spectrometry instruments to serve as the apparatus for the method. It allows a multi-functionality of the instruments, enabling them toperform both their original analytical function and the additional role of quality verification, thus optimizing laboratory operations.

[0014] Another one of those preferred further developments of the disclosed method comprise that the software applies specifications for the background fluorescence magnitude and variation across the well plate, specifically, standard deviation, or other metrics. This adds a level of specificity to the software's analysis capabilities, with the feature being the application of defined criteria for evaluating the background fluorescence. It includes the use of statistical measures such as standard deviation, to ensure a detailed and quantifiable assessment of the well plate's fluorescence properties, thereby enhancing the precision of the quality verification process.

[0015] Another one of those preferred further developments of the disclosed method comprise that the software provides a Pass or Fail result to a user, a report including a result tracking and / or a certificate of analysis. This emphasizes the software's ability to deliver an immediate and clear output of the analysis in the form of a Pass / Fail result. Additionally, it can provide detailed reports and certificates of analysis. The point of this feature lies in its support for traceability and documentation of quality control processes, which is particularly beneficial in regulated research environments.

[0016] Another one of those preferred further developments of the disclosed method comprise that the Pass or Fail result is tagged to an Identifier of the scanned well plate, forwarded to the control unit of the high-sensitivity optical spectrometry instrument, wherein the control unit initially denies the use of well plate with a tagged Fail result. This links the mentioned Pass / Fail result to a unique identifier for each well plate. The distinctive feature is the communication of this result to the instrument's control unit, which is programmed to automatically reject any well plate marked as Fail. It also prevents the inadvertent use of defective well plates in critical experimental procedures.

[0017] Another one of those preferred further developments of the disclosed method comprise that the report further includes several metrics of thebackground fluorescence, wherein limits are applied to the metrics of background magnitude and intra-well plate background uniformity. This further specifies the previously described reporting capabilities. The feature here is the inclusion of multiple background fluorescence metrics in the report, with applied limits to these metrics for both overall magnitude and uniformity. That enables the provision of comprehensive quality data, allowing for a more nuanced assessment of well plate performance.

[0018] Another one of those preferred further developments of the disclosed method comprise that the well plates are measured and individually verified at a 100% sampling rate, a lower sampling rate or at a utilized manufacturing lot sampling rate. That introduces a flexibility in the sampling rate at which well plates are verified, with the feature being the choice of verifying every well plate (100% sampling rate), a subset, or by manufacturing lots. It allows to accommodate varying levels of quality control rigor based on practical considerations or the criticality of the application.

[0019] Another one of those preferred further developments of the disclosed method comprise that the high-sensitivity optical spectrometry instrument is used for the detection of protein concentrations in a prepared assay solution. That specifies the invented method to the practical application of detecting protein concentrations in assay solutions using a high-sensitivity epi-fluorescence instrument. The feature is the direct connection between well plate quality and its suitability for use in sensitive protein assays, highlighting the importance of accurate background fluorescence measurement in such applications.

[0020] Another one of those preferred further developments of the disclosed method comprise that as high-sensitivity optical spectrometry instruments epi- 1 fluorescence microscopes, single molecule detection instruments, or Raman spectrometers are used. This specifies the range of compatible instruments that can employ the method to include optical spectrometry instruments but also in general fluorescence microscopes, single molecule detection instruments, or Raman spectrometers. This feature is about inclusivity, broadening the method's applicability to a variety of sensitive fluorescence-based detection instruments.

[0021] Another one of those preferred further developments of the disclosed method comprise that the maximum tolerance level will result in an increase in signal-to-noise ratio (SNR or CV) of no more than 4% at low concentrations. Since experimental studies and Monte Carlo simulations show that even high- quality well plates may have background variations which increase signal CV’s greater than 20%, thus severely affecting signal accuracy and precision, a maximum threshold of below 4% is absolutely sufficient for a suitable use of the respective well plates.

[0022] A further component of the claimed invention is a method to detect protein concentrations in a prepared assay solution by using a high-sensitivity optical spectrometry instrument wherein only well-plates are used whose background fluorescence has been confirmed to be within a given maximum tolerance level by applying a method as previously described. With the application of the previously described method, it is ensured that only well plates that have been verified to be within the maximum tolerance level of background fluorescence are used for detecting protein concentrations. This mandates that before protein concentration detection, well plates must be pre-verified using the method outlined in the preceding claims, thereby guaranteeing the reliability of the assay results.

[0023] A further component of the claimed invention is a system to measure and verify the background fluorescence of well plates for use with a high-sensitivity optical spectrometry instrument, comprising of a computer and an apparatus which is connected to the computer, wherein the system is configured to perform the method steps as previously described. That encapsulates a system that includes both the computer and a connected apparatus configured to perform the previously described method. The feature is about the integration of both hardware and software into a cohesive system designed to carry out automated quality verification of well plates, providing a streamlined solution for ensuring well plate integrity for high-sensitivity fluorescence analysis.BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

[0024] Figure 1 : showing an example of a test report

[0025] Figure 2: showing an image of an empty well plate inserted into the instrument (left) and an image of the well plate ‘shoe’ placed over top of the well plate (right).

[0026] Figure 3: showing the graphic user interface of the software loading a template.

[0027] Figure 4: showing the set of an output filename for the test case in the software.

[0028] Figure 5: showing the software with the Start button highlighted, and Load Plate prompt with correct selections.

[0029] Figure 6: showing the Plate Info text box with the filled in information for the lot # and plate ID.

[0030] Figure 7: showing the second page of the result report with well plate test result PASSING at the top of page.DETAILED DESCRIPTION OF THE INVENTION

[0031] The invention will be explained in more detail by presenting one preferred exemplary embodiment.

[0032] The invented method can be applied for different high-sensitivity optical spectrometry instruments, in particular single molecule detection (SMD) instruments as the newest SMCxPRO® and SMC® assay product line mentioned in the unpublished US Provisional application 63 / 605,656.

[0033] The invention is now further explained by one preferred embodiment, as described in the follows.

[0034] Described is a procedure or workflow disclosing how to execute the invented method on exemplary well plates which are used with the specific single molecule detection (SMD) instrument, in this embodiment a modified SMCxPRO. The instrument is speficially adjusted to only measure an empty well plate, measuring the background autofluorescence, called Noise parameter across 96 of this specific well plate’s wells. It doesn’t have to be 96 wells for the invention o work. More is more statistically accurate. Less may be fine too. It is a tradeoff in the process of time vs accuracy.

[0035] The instrument delivers a measurement of the average autofluorescence per well. Noise is basically a fraction of the total collection of photons per second. In a further embodiment this can also be scaled to energy / second (or power) - such as Joules / second (or Watt).

[0036] The so called xPRO software will be used to control the instrument. Another software, the so called Aurora QC Report Generator software will be used for analysis and interpretation of results. The usage of two separate softwares is not a specific feature of this invention, but just an embodiment. It is also possible that the instrument control software could do the analyzing part in another embodiment. But here the instrument and custom software will be setup prior to use. The Instrument shall be serviced every 6 months - upon which calibration and alignment will be verified.

[0037] The applied method consists of the following steps:

[0038] Step 1 - Insert Aurora well plate for testing into the instrument with the following substeps:• Insert Aurora plate into the instrument.• Open the instrument cover using xPRO software• Place plate into plate carrier with A1 corner oriented to the top left• Plate should be empty• Plate ‘shoe’ is placed over top of plate after inserted. This blocks the effect of stray light.

[0039] Figure 2 shows an Image of this step with the empty Aurora plate inserted into instrument (left) and an image of the plate ‘shoe’ placed over top of the plate (right).

[0040] Step 2 - Open a scan template ‘Q1-FastScan.xpt’

[0041] In the software it has to be ensured that the template ‘Q1 -FastScan. xpt’ is loaded. Figure 3 shows an Image of the template ‘Q1 -FastScan. xpt’ loaded into the software.

[0042] Step 3 - Set filename and save directory

[0043] In the software, set a descriptive filename and set the save directory.

[0044] Figure 4 shows an image of the output filename set in the software.

[0045] Step 4 - Scan plate

[0046] Sub steps:• In the software, select ‘Start’ to begin scanning plate.• Ensure that Aurora plate type is selected.• Scan mode is Standard.• Skip thermal stabilization

[0047] Figure 5 shows an image with the software's GUI with the Start button highlighted, and Load Plate prompt with correct selections.

[0048] Step 5 - Run software program: Aurora QC Report Generator

[0049] After the scan has finished, the resulting XPR file will be analyzed using the Aurora QC Report Generator software. To do so in this preferred embodiment, this software needs to be started and the XPR file has to be loaded by the software. For this a text box in the GUI of the Aurora QC Report Generator software needs to be filled out including the lot #, an ID for the plate (i.e. Case 43, Plate 1 ). The report filename will default to the same filename as the CSV - and shouldn’t require changing.

[0050] Figure 6 shows a respective GUI screenshot with the Plate Info text box, the filled out information for the lot # and plate ID. The report filename should not require any change.

[0051] When the results of the average fluorescence from the 96 wells is fed into the software to calculate the average autofluorescence of all the wells across the plate and the variation of the fluorescence in wells across the plate. A clear PASS / FAIL result of a good versus bad well plate is based on the average fluorescence being below a certain threshold value, i.e. Noise must be lest than 6, AND the fluorescence variation being below a certain value, i.e. Noise CV < 5%.

[0052] To do so a progress bar will indicate the application is analyzing the XPR file. After analysis is complete (~30 seconds), a PDF report will generate automatically in the same directory as the XPR file.

[0053] The PDF report may automatically open depending on configuration settings setup upon software installation.

[0054] Navigate to page 2. A clear indication of Pass or Failure will be provided along with experimental details and primary summary statistics. The remainder of the report contains visual heat maps and plots of various plate parameters.

[0055] Figure 7 shows the mentioned Page 2 of the PDF report. In this case showing plate PASSING at top of page. The most important statistics are highlighted at the bottom of the page, like the fluorescence magnitude (Noise Avg) and variation (Noise CV).

[0056] A Certificate of Analysis for each manufactured lot should denote PASS / FAIL rate for all samples. All measurements from each sample in the lot should also be recorded.

[0057] Screenshots of another example measurement report showing Pass / Fail message, summary statistics, and well plate background heatmap are shown in Figure 1 . Note that plate fluorescence background is called ‘Noise’ in this report.

[0058] With the PASS or FAIL result the measured well plate can the be tagged as ready for use in real measurement with a high sensitivity optical instrument in case of a PASS result or tagged to be sorted out, in case of a FAIL. So it is ensured that only suitable well plates with a low background fluorescence which do not influence the high sensitivity optical instrument will be used.

[0059] For the SMC® product line, background specifications have been developed to optimize performance versus well plate costs. Current specifications provide limits on plate background magnitude and uniformity. Compared to a ‘perfect’ or ‘gold standard’ well plate, these specifications limit the quality to a level that will result in an increase in signal CVs of no more than 4% at low concentrations.

Claims

CLAIMS1. A Method to measure and verify the background fluorescence of well plates for use with a high-sensitivity optical spectrometry instrument via a computer, the following steps comprising:• Inserting the well plate into an apparatus which is connected to the computer and applies the scan method and data collection of the high-sensitivity optical spectrometry instrument;• Forwarding the collected data to the computer where a software analyzes the data to determine the background fluorescence of the scanned well plate and checks if the background fluorescence exceeds a given maximum tolerance level;• Sorting out the well plate if the given maximum tolerance level has been exceeded.

2. The Method according to claim 1 , wherein the application of the scan method and data collection is performed either at a manufacturer, inventory, or QC site prior to the delivery of the well plate to a customer.

3. The Method according to claim 1 , wherein the application of the scan method and data collection is performed at the customer location in the customer instrument in an automated form.

4. The Method according to claim 1 , wherein as apparatus a Custom Hardware is used which is designed or modified from an existing platform.

5. The Method according to claim 1 , wherein as apparatus a modified high-sensitivity optical spectrometry instrument is used.

6. The Method according to claim 1 , wherein the software applies specifications for the background fluorescence magnitude and variation across the well plate, in particular a standard deviation, or other metrics.

7. The Method according to claim 1 , wherein the software provides a Pass or Fail result to a user, a report including a result tracking and / or a certificate of analysis.

8. The Method according to claim 7, wherein the Pass or Fail result is tagged to an Identifier of the scanned well plate, forwarded to the control unit of the high-sensitivity optical spectrometry instrument, wherein the control unit initially denies the use of well plate with a tagged Fail result.

9. The Method according to claim 7, wherein the report further includes several metrics of the background fluorescence, wherein limits are applied to the metrics of background magnitude and intra-well plate background uniformity.

10. The Method according to claim 1 , wherein the well plates are measured and individually verified at a 100% sampling rate, a lower sampling rate or at a utilized manufacturing lot sampling rate.11 . The Method according to claim 1 , whereinthe high-sensitivity optical spectrometry instrument is used for the detection of protein concentrations in a prepared assay solution.

12. The Method according to claim 1 , wherein as high-sensitivity optical spectrometry instruments epi- I fluorescence microscopes, single molecule detection instruments, or Raman spectrometers are used.

13. The Method according to claim 1 , wherein the maximum tolerance level will result in an increase in signal-to- noise ratio (SNR or CV) of no more than 4% at low concentrations.

14. A Method to detect protein concentrations in a prepared assay solution by using a high-sensitivity optical spectrometry instrument wherein only well-plates are used whose background fluorescence has been confirmed to be within a given maximum tolerance level by applying a method according to claims 1 to 12.

15. A System to measure and verify the background fluorescence of well plates for use with a high-sensitivity optical spectrometry instrument, comprising of a computer and an apparatus which is connected to the computer, wherein the system is configured to perform the method steps of claims 1 to 13.

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