Time-division multiplex scan methodology

Abstract

This invention disclosure presents a novel method and system for protein detection that significantly enhances accuracy by minimizing cross-excitation and cross-emission effects commonly encountered in multiplex fluorescence assays. The method involves isolating multiple proteins from a sample, labeling them with fluorescent inks, and arranging them on a sample substrate such that no adjacent labels have overlapping excitation or emission spectra. Each labeled protein is sequentially excited by a specifically matched laser wavelength and detected by a corresponding detector, equipped with adjustable optical filters, to ensure precise measurement of the fluorescence emission. The system comprises a high-sensitivity optical detection instrument that includes a light source, a detector, and a control unit. The control unit is designed to adjust the sequence of excitation and detection based on the detected fluorescence intensity, optimizing the dynamic range of the assay.This methodology and system setup allow for high-throughput protein detection with reduced spectral interference, providing a more reliable and efficient approach to quantifying multiple proteins in complex samples.

Description

Time-division multiplex scan methodologyBACKGROUND OF THE INVENTION

[0001] The disclosed invention relates to a method and a system for protein detection via fluorescence detection wherein the excitation and emission detection for each target label are performed sequentially and the target labels are arranged beside each other in a specific way to avoid cross-talk.

[0002] The invention belongs to the technical fields of fluorescence based Protein Detection.

[0003] The detection of proteins is a fundamental aspect in various fields such as biotechnology, medicine, and pharmaceutical sciences. Instrumentation for protein detection is diverse, incorporating several methodologies that have evolved to address specific needs:

[0004] 1 . Western Blot: A legacy method for protein identification that is semi- quantitative and commonly used for screening purposes.

[0005] 2. ELISA (Enzyme-Linked Immunosorbent Assay): A pivotal technique in the industry, known for its quantitative analysis with a typical sensitivity of 10 pg / mL. It is generally used in a single-plex format.

[0006] 3. Bead-Based Sandwich ELISA (e.g., Luminex): This method allows for high multiplexing potential, accommodating up to 80 markers with low sample volumes. It is both quantitative and used for screening, with a sensitivity ranging approximately from 1 to 10 pg / mL.

[0007] 4. Immunoassay qPCR (e.g., ProQuantum): Offers highly sensitive quantitative detection in a single-plex format, with sensitivity levels as low as 0.01 pg / mL.

[0008] 5. Immunoassay Single Molecule Detection (e.g., SMCxPRO®): Utilizes single plex and 2-Plex formats for ultra-sensitive quantitative analysis. The 2-Plexcapability is facilitated by the optical architecture disclosed in this document, with sensitivities reaching as low as 0.01 pg / mL.

[0009] 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 lense for coupling the fluorescent light to an optical fiber, at least one fiber patch cables to couple fluorescently focused light to at least 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 lense, 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.

[0010] Most protein detection methods, with the exception of chemiluminescent ELISAs, utilize fluorescent labels. Fluorescence detection employs a specific wavelength light source, typically a laser, for excitation, and a photodetector equipped with optical filters to measure the fluorescence spectrum of the label. Modem fluorescence detection platforms support multiplexed detection but face several challenges.

[0011] First is the Cross-Excitation Effects: An unintended excitation of nontarget labels, such as excitation of AF750 by a 647 nm laser, which is intended for AF647.

[0012] Second the Cross-Emission Effects, whic are an opverlap of non-target label emissions into the target detection band, for example, emissions from AF647 leaking into the detection band designated for AF750.

[0013] Together they form crosstalk from Cross-Excitation and Cross-Emission, which results in overlapping emissions from non-target labels with target labels, necessitating complex 'unmixing' procedures to subtract or remove this overlap, thus avoiding inaccurate measurements.

[0014] Both cross-excitation and cross-emission challenge accurate detection, since a single detector may measure signals from multiple (mixed) target labels (that are indistinguishable). Methods of ‘unmixing’ may include:

[0015] First single color controls with Dilutions of fluorescent labels, typically measured prior to a scan, to enable calculation of spectral overlap and development of a correction matrix. This correction matrix enables subtraction or ‘unmixing’ of cross-talk in data.

[0016] And second non-negative matrix factorization, wherein an automatic calculation of correction matrix directly from the scan data with some assumption on theoretical spectra.

[0017] Such unmixing methodologies and algorithms can work well but present additional workflow complications as well as waste precious samples.Additionally, crosstalk can never be fully un-mixed and will always lead to accuracy degradation.

[0018] The task of this invention is therefore to disclose a new method and a system for protein detection which dimishes cross-talk but avoids the known problems of the state of the art.BRIEF SUMMARY OF THE INVENTION

[0019] This task can be solved by a method for protein detection wherein multiple proteins are isolated from a sample, labeled with a fluorescent ink beside each other on the sample as a target for detection, each resulting target label is then first excited with a respective light source with a specific wavelength resulting in an excitation line which causes a fluorescence emission from the target label, and then sampled with a detector regarding a small band of the label’s fluorescence spectrum, wherein the excitation and emission detection for each target label are performed sequentially and the target labels are arranged beside each other in such a way that only protein targets labelled with fluorescent ink wherein either the excitation or the emission band or both are not overlapping are arranged adjacent to each other. This setup ensures that each labeled protein can be individually excited and its fluorescence detected without interference from adjacent labels. By arranging the labels so that their excitation or emission bands do not overlap adjacently, the method reduces spectral overlap, enhancing the detection's accuracy and specificity. It is important that both central features, the sequential excitation and emission monitoring for each target label and the respective label arrangement, are performed together. If only the label arrangement would be done but still with a multiplex monitoring the cross-talk could not be avoided. And a sequential protein detection monitoring without arranging the labels in the described way would either also lead to crosstalk, since otherwise arranged adjacent labels which have both interfering crossexcitation and cross-emission will also result in cross-talk or in case of no further adjacent labels being placed on the sample the whole detection procedure would become extremely inefficient.

[0020] Advantageous and therefore preferred further developments of this invention emerge from the associated sub claims and from the description and the associated drawings.

[0021] One of those preferred further developments of the disclosed method comprise that as light sources with a specific wavelength a laser with a wavelength suitable to the respective target label is used. It means further that foreach different wavelength another laser needs to be used. By matching the laser wavelength to the optimal excitation wavelength of each fluorescent label, this feature maximizes the efficiency of fluorescence excitation. This targeted excitation is essential for achieving strong and distinct emission signals, thereby improving the sensitivity and specificityof the detection.

[0022] Another one of those preferred further developments of the disclosed method comprise that for a test setup with at least four target labels as light sources with a specific wavelength a system of at least four lasers is used that satisfies the property of “mirror symmetry” for excitation and emission. This specific setup is designed to further minimize cross-talk and is kind of a trade-off between minimum cross-talk and acquisition time for such 4-plex system. The fou lasers in this system can be for example, green and NIR as ONE laser being on together with collection filter for Green and NIR with minimum (or zero) cross-talk, while sequenced with another group, RED & IR lasers for collecting another two analytes simultaneously.

[0023] Another one of those preferred further developments of the disclosed method comprise that as detector a photodetector, avalanche photodiode, or photomultiplier tube is used. This flexibility allows the selection of a detector technology that best fits the sensitivity and speed requirements of the assay. Different detection technologies offer various advantages in terms of response time, sensitivity to low light levels, and noise performance, making this choice critical for optimizing assay performance.

[0024] Another one of those preferred further developments of the disclosed method comprise that an immunoassay single molecule detection instrument comprising the light source, the detector and a control unit is used. This integration facilitates a streamlined and cohesive operation, reducing complexity in setup and operation. It ensures that all components are optimized to work together, which can enhance the reliability and reproducibility of the protein detection process. The control unit can also be used to program the instrument to perform the respective method steps of excitation, emission monitoring and even data anylses of the monitored data. In case the control unit is not suited to do so,or in cases no integrated single molecule detection instrument is used, this part can also be done by an external computer which receives the monitored detection data.

[0025] Another one of those preferred further developments of the disclosed method comprise that single-color controls data is collected by the control unit for monitoring the immunoassay single molecule detection operation and / or for generating a cross-talk correction matrix. Despite the invented approach avoiding cross-talk efficiently, there might be use cases where it is still beneficial to generate and apply a cross-talk correction matrix. This can be done in an additional method step.

[0026] Another one of those preferred further developments of the disclosed method comprise that the control unit is configured to automatically adjust the sequence of excitation and detection based on the detected fluorescence intensity to optimize the assay's overall dynamic range. This adaptive feature allows the system to dynamically optimize its settings based on the actual fluorescence signals received, which can vary significantly between samples. This adaptability helps in maintaining optimal detection conditions throughout the assay.

[0027] Another one of those preferred further developments of the disclosed method comprise that the detector uses adjustable and / or exchangeable optical filters to sample the target label’s fluorescence spectrum. This customization enables the detector to be specifically tuned to the emission spectra of the fluorescent labels used, which is critical for reducing detection of stray light or fluorescence from non-target labels, thereby enhancing the purity of the detection signal.

[0028] Another one of those preferred further developments of the disclosed method comprise that the excitation wavelengths and detection bands are selected based on the known spectral properties of the target labels. While obviously both the excitation wavelengths and the detection bands have to be in wavelength ranges where target labels actually can be excited and emitfluorescence light, it is preferable to select the most suitable range for the procedure. That might not always be the wavelength with the highest excitation sensitivity and / or emission efficiency, in particular if these wavelengths are overlapping too much with the values from the adjacent target labels and therefore not fulfill the requirements regarding the target label arrangement.

[0029] Another solution to the given task is a target label layout for protein detection target labels wherein the target labels are arranged beside each other in such a way that only target labels with proteins whose respective excitation or emission bands or both are not overlapping are arranged adjacent to each other. This layout design prevents adjacent labels from having overlapping excitation or emission spectra, crucial for reducing cross-talk between labels. Such spatial arrangement is especially important in high-throughput assays where many labels are used on one sample.

[0030] A further solution to the given task is a System for protein detection, comprising multiple protein detection target labels as previously described, a light source with a specific wavelength, a high-sensitivity optical detection instrument, and a control unit wherein the system is configured to perform the previously described method steps.

[0031] These preferred further developments collectively describe a sophisticated protein detection method and system designed to maximize detection accuracy by carefully controlling the excitation and detection of fluorescently labeled proteins, thereby reducing potential errors caused by spectral overlap and enhancing the overall performance of the assay.BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

[0032] Figure 1 : showing an example of a test report

[0033] Figure 2: showing an image of AF647 and AF750 excitation functions, with common excitation lines.

[0034] Figure 3: showing an image of AF647 and AF750 emission, with common detection bandsDETAILED DESCRIPTION OF THE INVENTION

[0035] The invention will be explained in more detail by presenting one preferred exemplary embodiment.

[0036] This preferred exemplary embodiment of carrying out the invention involves implementing a protein detection system designed to minimize crossexcitation and cross-emission effects using a sequential scan methodology. This example will detail how to execute the invention using the specific conditions and configurations that optimize the performance of the protein detection system, as described in the invention and illustrated by Figures 1 , 2 and 3.

[0037] First about the Equipment Setup:

[0038] As light source with two lasers are used, where the first laser operates at a wavelength of 642 nm and the second at 730 nm.

[0039] As detectors two high-sensitivity detectors equipped with optical filters are used, with the first detector having a detection band of 680 / 40 nm and the second detector having a detection band of 780 / 40 nm.

[0040] Furthermore an immunoassay single molecule detection instrument (SMCxPro) is used that integrates the lasers, detectors, and a control unit capable of handling the sequential operation and respective data processing.

[0041] Then two fluorescent target labels, AF647 and AF750, are used, which are known for their distinct excitation and emission properties as shown in Figures 2 and 3.

[0042] To prepare those target labels multiple proteins are isolated from a sample and labeled with either AF647 or AF750. The proteins labeled with AF647 and AF750 are arranged on the target such that their respective excitation and emission bands do not overlap adjacently.

[0043] The invented method itself consists of several method steps which are shown in Figure 1 and described in more detail in the follows:

[0044] First step: First Laser Activation (642 nm) at the AF647 labeled proteins.

[0045] For the procedure the 642 nm laser is activated to excite the AF647 labels. The fluorescence emission is monitored with the detector using the 680 / 40 nm detection band. Since the excitation of AF647 does not effectively excite AF750, there is only minimal cross-excitation.

[0046] The emitted fluorescence from AF647 is detected, and any minor fluorescence from AF750 due to cross-excitation is negligible because AF750 does not emit within the detection band of 680 / 40 nm. And any emission to AF750 channel from cross-excitation is thrown away before second laser activation.

[0047] Next step: Second Laser Activation (730 nm) at the AF750 labeled proteins. After completing the scan with the first laser, the 730 nm laser is activated to excite the AF750 labels. The fluorescence emission is monitored using the detector for the 780 / 40 nm detection band. The emitted fluorescence from AF750 is detected, and since AF647 is not excited by the first laser with 642 nm consequently there is no emission in this detection band and therefore no cross-emission interference.

[0048] If a multiplex observation with both lasers exciting at the first time would have been performed, both cross-excitation and cross-emission would have been inevitable, as can be seen in Figures 2 and 3. The single-plex approach plus arranging only labels adjacent to each other where either the excitation or the emission band or both are not overlapping ensures that cross-excitation and cross-emission can effectively avoided. Of course it would be also possible to simply monitor only one protein labelled with one ink in a target on a sample, but that would be greatly time- and cost-ineffective.

[0049] Although the sequential scanning as described above avoids cross-talk, any residual interference detected can still be further optimized corrected in anoptional step of using a pre-determined correction matrix based on baseline scans of single-labeled controls.

[0050] In the last method step the fluorescence intensities for each label are then quantified, and protein concentrations are calculated based on these intensities.

[0051] This preferred embodiment realizes the ‘x-axis’ symmetry of the excitation and emission functions for each label as shown in Figures 2 and 3. It will be explained here using the provided preferred embodiment.

[0052] First considering the AF647 label. The “long tail” of the excitation band points to the left, meaning the shorter wavelengths, and thus has no interference with the 2nd excitation line. The ‘long tail’ of the emission band points to the right, the longer wavelengths, i.e. the x-axis symmetry, and thus does interfere with detection band 2.

[0053] The ‘tail directions’ for the AF750 label point in the same directions as AF647 discussed above. This will hold for most traditional labels, due to the quantum / electronic nature of the excitation. However, the result is opposite compared to the AF750 dye - cross-excitation interference with excitation line 1 but no cross-emission to detection band 1 .

[0054] This “mirror symmetry” of the excitation and emission bands results in a kind of mirror symmetry of the cross-excitation and cross-emission of two adjacent dye labels and is fundamentally how this methodology works.

[0055] This methodology can be expanded to larger numbers of PLEXED labels, as long as their spectral separation allows the characteristics just discussed between any two adjacent labels. Therefore, in a further preferred embodiment of the invention it is expandable to multiplex with more than two channels or lasers (analytes). One example is a FQ system wherein the one laser is actually designed as one group of laser that satisfies the property of “mirror symmetry” of excitation and emission, for example, green and NIR as ONE laser being on together with collection filter for Green and NIR with minimum (or zero) cross-talk, while sequenced with another group, RED & IR lasers for collecting another twoanalytes simultaneously. This results in an optimal trade-off between minimum cross-talk and acquisition time for 4-plex system.

[0056] Altogether, this sequential scanning method in all disclosed preferred embodiments, as implemented in the described setup, effectively avoids the issues of cross-excitation and cross-emission, leading to more accurate and reliable protein quantification. The methodology leverages the inherent spectral properties of the AF647 and AF750 dyes, as demonstrated in Figures 2 and 3, to ensure that each label's signal is distinctly measured without interference, thereby optimizing the assay’s performance.

Claims

CLAIMS1 . A Method for protein detection wherein multiple proteins are isolated from a sample, labeled with a fluorescent ink beside each other on the sample as a target for detection, each resulting target label is then first excited with a respective light source with a specific wavelength resulting in an excitation line which causes a fluorescence emission from the target label, and then sampled with a detector regarding a small band of the label’s fluorescence spectrum, wherein the excitation and emission detection for each target label are performed sequentially and the target labels are arranged beside each other in such a way that only protein targets labelled with fluorescent ink wherein either the excitation or the emission band or both are not overlapping are arranged adjacent to each other.

2. The Method according to claim 1 , wherein as light sources with a specific wavelength a laser with a wavelength suitable to the respective target label is used.

3. The Method according to claim 1 , wherein for a test setup with at least four target labels as light sources with a specific wavelength a system of at least four lasers is used that satisfies the property of “mirror symmetry” for excitation and emission.

4. The Method according to claim 1 , wherein as detector a photodetector, avalanche photodiode, or photomultiplier tube is used.

5. The Method according to claim 1 , wherein an immunoassay single molecule detection instrument comprising the light source, the detector and a control unit is used.

6. The Method according to claim 5, wherein single-color controls data is collected by the control unit for monitoring the immunoassay single molecule detection operation and / or for generating a cross-talk correction matrix.

7. The Method according to claim 5, wherein the control unit is configured to automatically adjust the sequence of excitation and detection based on the detected fluorescence intensity to optimize the assay's overall dynamic range.

8. The Method according to claim 1 , wherein the detector uses adjustable and / or exchangeable optical filters to sample the target label’s fluorescence spectrum.

9. The Method according to claim 1 , wherein the excitation wavelengths and detection bands are selected based on the known spectral properties of the target labels.

10. A target label layout for protein detection target labels wherein the target labels are arranged beside each other in such a way that only target labels with proteins whose respective excitation or emission bands or both are not overlapping are arranged adjacent to each other.

11. A System for protein detection, comprising multiple protein detection target labels according to claim 10, a light source with a specific wavelength, a high-sensitivity optical detection instrument,and a control unit wherein the system is configured to perform the method steps of claims 1 to 9.

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