Patent Application
20230384314
The instant application contains a Sequence Listing which has been submitted in .txt format via EFS-Web and is hereby incorporated by reference in its entirety. Said txt copy is created on Oct. 18, 2021, named “Sequence Listing 95062105428 ST25”, and is 2,266 bytes in size.
The present disclosure relates to a method for bio-analysis of complex samples and enabling biomolecule detection using a combination of electrochemical detection electronically coupled with additional downstream methods including optical detection, polymerase chain reaction (PCR) methods, gene sequencing methods, immunoassays, and mass spectrometric (MS) detection technologies as additional analysis. The detection of biomolecule and cells in whole blood, serum, plasma, urine, wound fluid, bronchial lavage, and other complex biological samples is improved in the disclosed. This disclosure expands the compatibility of electrochemical detection with other bio-analysis methods.
Bio-analysis by combining results from multiple methodologies, namely electrochemical, optical, imaging, genetic, and mass spectrometric analysis, is becoming increasing more common for measurements of complex in-vitro, cell and tissue samples. Additionally, new mass spectrometric (MS) methods using mass labels to perform biomarker analysis are becoming increasingly more competitive and common, with noted ability to analyze rare biomolecules of interest at picomolar (pM) sensitivity. Such new methods lead to discovery of new masses, offering of high sample through-put, improved specificity, and/or multiplexed detection of multiple analytes in a single analysis.
New mass spectrometric (MS) methods using mass labeling are used commonly, such as the Isotope-Coded Affinity Tags (ICAT) method, Tag for Relative and Absolute Quantitation (iTRAQ), and Tandem Mass Tag (TMT) for mass spectrometric proteomic analysis. These methods of attaching a mass label to the biomolecule to be measured all suffer from contamination, expansive reagents, the use of isotopes as labels, and overlapping masses. These methods are also limited by the size and fragmentation of the analyte to be measured. Utilizing mass labels in mass spectrometric immunoassays (MS-IA) has improved the analysis by eliminating the need to purify the biomolecule before the analysis, by eliminating the need to chemically attach the mass label to the biomolecule, by eliminating the need for trypsin to fragment biomolecules, and the need for isotope labeling. A mass labeling approach for MS-IA utilizing a metal attached to antibodies and detecting the presence of metal by MS has been successfully used after optical imaging of cells and tissues. (Bandura 2009, Lee 2008). However, this requires expensive and specialized mass analyzers for metal detection.
Recently, the Signal Ion Emission Reactive Releasee Amplification (SIERRA) method for MA-IA (as described in, for example, Pugia U.S. Pat. Nos. 10,809,264, 11,061,035, Analytical Chem 2016, 2019, 2021) was developed using releasable organic compounds as the mass label attached to the affinity agents. These mass labels can be detected on almost any mass spectrometer (MALDI, ESI-MS-MS, Triple Quad) and eliminate the issues noted above. An alteration agent, namely, an acid or reducing agent, is used to break a linkage bond and release the mass label from the antibody for mass analysis. Detection of biomolecules in cells was demonstrated after optical imaging of cells with little background for MS-IA from complex samples (as described in, for example, Pugia Anal Chem 2016, 2019).
It is highly desirable that a rapid point of care (POC) method, such as electrochemical immunoassays (EC-IA), be compatible with the additional methods such as mass spectrometric (MS), optical imaging, and polymerase chain reaction (PCR) methods. Added flexibility of allowing the sample to be analyzed multiple times with these separate methods provides additional information and allows for laboratory re-testing of an analyte captured from a sample at POC. The SIERRA MS-IA has been shown to be compatible with optical fluorescence labels and to be non-destructive to DNA and RNA, allowing polymerase chain reaction (PCR) methods (as described in, for example, Pugia Anal Chem 2016, 2019, 2021). However, the electrochemical immunoassays (EC-IA) had to be run separately as electrochemical reactions during the electrochemical detection method due to the potential damage to the SIERRA reagent.
Previously, Pugia US2018/0284124 demonstrated that electrochemical reduction can break cleavable X-Y linkages of SIERRA reagents and that oxidation reactions can cause addition of phenols and amines to aromatic groups, peptides, proteins, and other biomolecules. Horseradish peroxidase (HRP) or alkaline phosphatase (ALP) are commonly used for electrochemical immunoassays (EC-IA) and are known to cause addition of phenolic and phenyl-amines substrates to aromatic amino acids. For example, immunoassays based on ALP and HRP enzymes such as Tyramide Signal Amplification (TSA™) (Pugia WO2015184144) cause covalent attachment of phenols and phenyl amines of the substrates to the peptide tyrosine groups.
PCT/US2020/055931 (hereinafter the “IBRI PCT”), which is incorporated by reference in its entirety, has recently demonstrated a device format that could perform multiple analysis of biomolecules directly on complex samples using multiple analyte detection microwells for electrochemical detection of target analytes. The analyte detection microwell includes a size exclusion filter with one or more pores, an electrochemical detector, and affinity agents for target analyte capture and detection which operate under a low hydrodynamic force. The affinity agent for detection is attached to a reagent capable of generating an electrochemical label. The affinity agent for capture is attached to a reagent capable of binding a surface in the microwell. The electrochemical label is detected by a working electrode and a reference electrode placed in the microwell to measure the label formed by the affinity agent for detection. The format enables capture of biomolecules and immunoassay detection of captured biomolecules in a convenient format without user intervention, with the added benefit of being able to remove and store the analyte detection microwell for additional downstream analysis.
The IBRI PCT device can be used with reagents for affinity assays such as electrochemical immunoassay (EC-IA), optical imaging, polymerase chain reaction (PCR) methods, mass spectrometric immunoassays (MS-IA), and mass spectrometric proteomics methods of the biomolecules captured on the analyte detection microwell. Descriptions of these assays utilized may be found in Pugia, M. J. et al., “Multiplexed SIERRA Assay for the Culture-Free Detection of Gram-Negative and Gram-Positive Bacteria and Antimicrobial Resistance Genes” Anal Chem, 2021 (published after provisional application). In one example, polyclonal affinity reagents were used as a sandwich assay pair by placing an affinity label (biotin) on some of the polyclonal antibody and placing an electro-chemical generating catalyst on the remaining polyclonal antibody. This format allows the biomolecules to be immediately captured on the filtration membrane using neutravidin. For multiplexed analysis, the filtration membrane is divided into multiple micro-wells with the size exclusion filter bottom.
While technologies capable of performing additional analysis of detection of rare molecules and cells after POC analysis exist, the field still requires improvements of electrochemical analysis methods to be compatible with mass spectrometric (MS) detection and other methods to allow simultaneous addition of all reagents in one sample processing step, and to allow performing an electrochemical analysis prior to mass analysis. A solution to this problem is the subject of this invention.
An object of a non-limiting embodiment of the present disclosure is to provide an electrochemical analyte detection method of target analytes wherein said detection methods do not interfere with downstream detection where reagents can be added simultaneously and used in downstream analysis. This allows additional reagents to be included as mass reporters serving analyte standards for measuring analyte and demonstrating analyte integrity and identity. Analyte integrity and identity are used to allow electronical coupling of electrochemical analyte detection to the downstream optical, genetic, or mass spectrometric results. Mass reporters and reagents for electrochemical are added simultaneously to specific analyte detection microwells, where an affinity agent can capture mass reporter, analytes, and reagents used for electrochemical signal generation. In non-limiting embodiments or examples, the affinity agent is additionally labeled with the label for optical detection or mass labels for mass spectrometric immunoassays.
In non-limiting embodiments, the reagents for electrochemical, mass reporters, optical, and mass spectrometric detection are processed together in a common electrochemical sensor microwell with a size exclusion filter, electrodes, and affinity agents for analyte capture reagent and analyte detection.
In non-limiting embodiments, affinity agents used for analyte capture allow the capture of cell, virus, particles or biomolecules as analytes.
In non-limiting embodiments, mass reporters of product and sample integrity are included and capable of producing analyte identity and integrity as indication of suitability of results for electronically electrochemical results coupling to downstream analysis results.
In non-limiting embodiments, downstream analysis methods include optical methods, genetic methods such as polymerase chain reaction (PCR), molecular probes and sequencing, immunoassays, and mass spectrometric (MS) detection technologies.
Further non-limiting embodiments or examples are set forth in the following numbered clauses.
Clause 1: A method of analysis of complex samples comprising: introducing an affinity agent with an attached catalyst capable of forming an electrochemical signal; and measuring the electrochemical signal that is capable of being electronically coupled to results of downstream methods.
Clause 2: The method of clause 1, wherein the method contains no interference with optical, genetic, or mass spectrometric detection.
Clause 3: The method of any of clauses 1-2, wherein a mass reporter measures the integrity and identity of an analyte by downstream methods.
Clause 4: The method of any of clauses 1-3, further comprising processing reagents for downstream methods in a common electrochemical sensor microwell with a size exclusion filter, electrodes, and affinity agents for analyte capture reagent and analyte detection.
Clause 5: The method of any of clauses 1-4, wherein electrochemical signals are generated upon oxidation at non-reducing voltages of >-0.1 and do not produce acidic pH.
Clause 6: The method of any of clauses 1-5, wherein mass reporters are generated upon reduction at non-reducing voltages of <−0.1.
Clause 7: The method of any of clauses 1-6, wherein the mass reporter causes production of the optical, genetic, or mass spectrometric results as an analyte.
Clause 8: The method of any of clauses 1-7, wherein a mass reporter measurement allows electronical coupling of electrochemical analyte detection results to downstream methods results.
Clause 9: The method of any of clauses 1-8, further comprising introducing a catalyst, wherein the catalyst is an enzyme to generate the electrochemical signal.
Clause 10: The method of any of clauses 1-9, further comprising introducing a catalyst, wherein the catalyst is a nanoparticle to generate the electrochemical signal.
Clause 11: The method of any of clauses 1-10, wherein an affinity agent contains a fluorescent label for optical detection.
Clause 12: The method of any of clauses 1-11, further comprising detecting a mass, wherein the mass reporters comprises analytes and/or labels which can be peptides, proteins, genes or small biomolecules.
Clause 13: The method of any of clauses 1-12, further comprising introducing mass reporters to a microwell, wherein an analyte is released for detection by additional downstream methods analysis.
Clause 14: The method of any of clauses 1-13, wherein an affinity reagent captures mass reporters, cells, and analytes.
Clause 15: The method of any of clauses 1-14, further comprising analyzing captured analytes by downstream analysis methods, wherein the downstream analysis methods comprise downstream optical methods, mass spectrometric methods, immunoassay methods, and genetic methods.
These and other features and characteristics of the present disclosure, as well as the methods of operation and functions of the related elements of structures and the combination of parts, will become more apparent upon consideration of the following description and the appended claims with reference to the accompanying drawings, all of which form a part of this specification, wherein like reference numerals designate corresponding parts in the various figures. It is to be expressly understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limits of the disclosure.
For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the embodiments illustrated in the drawings, wherein like reference numbers correspond to like or functionally equivalent elements, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications in the described embodiments, and any further applications of the principles of the invention as described herein, are contemplated as would normally occur to one skilled in the art to which the invention relates. Certain embodiments of the invention are shown in detail, but some features that are well known, or that are not relevant to the present invention, may not be shown for the sake of conciseness and clarity.
For purposes of the description hereinafter, the terms “end,” “upper,” “lower,” “right,” “left,” “vertical,” “horizontal,” “top,” “bottom,” “lateral,” “longitudinal,” “forward,” “reverse” and derivatives thereof shall relate to the example(s) as oriented in the drawing figures. However, it is to be understood that the example(s) may assume various alternative variations and step sequences, except where expressly specified to the contrary. It is also to be understood that the specific example(s) illustrated in the attached drawings and described in the following specification are simply exemplary examples or aspects of the invention. Hence, the specific examples or aspects disclosed herein are not to be construed as limiting. Moreover, as used in the specification and the claims, the singular forms of terms include plural referents unless the context clearly dictates otherwise.
For purposes of the description hereinafter, an analyte detection microwell used for electrochemical detection of target analytes is described in accordance with the IBRI PCT (
A non-limiting embodiment of the present disclosure is to provide an electrochemical analyte detection method of target analytes wherein said detection method does not interfere with downstream analysis methods. optical, genetic, or mass spectrometric detection.
The affinity agent can be additionally labeled with the mass labels for MS detection. In practice, the invention can make use of the same immunoassay reagent methods used for the electrochemical immunoassay (EC-IA) and SIERRA mass spectrometric immunoassays (MS-IA) as previously described as an example (Pugia Anal Chem 2017, 2019, 2021). These reagent methods can collect a sample and analyze the sample initially by reporting EC-IA results, which are discussed in Pugia et al. 63/006,833, 63/089,286, and 63/089,308 and incorporated herein by reference in their entireties. These reagent methods can electrochemically generate a signal as current in μA plotted against the voltage (V) for the electrochemical reporter captured by a high-affinity biotin onto a neutravidin linked to the size exclusion filter in a microwell with electrode.
The following
Referring to
Referring to
Referring to
One or more other microwells (5) are used for only analyte detection by electrochemical or mass spectrometric mass label detection as shown in
To demonstrate the invention, samples were simultaneously processed in an analyte detection microwell with mass reporters by the system, as shown in
coli (MyBioSource, San Diego, CA, USA), K. pneumoniae (Thermo Fisher
pneumoniae (Thermo Fisher Scientific) and P. aeruginosa (Abcam,
Method of Making Microwell Sensors with High-Affinity Capture Surface
Micro-filtration sensors (21) with arrays of microwells (5) of either 110 or 200 μm diameters and 300 μm depths were made using standard microfabrication photolithography techniques with <0.1 μm dimensional tolerance. Microwells (5) were patterned with the arrays inside a 6.5-mm diameter of 35 mm 2 or the size of a conventional ELISA plate well. In brief, film layers (4 to 20 μm) of dense, high-quality thermal SiO2 were patterned with a slotted pore (9.0×21.0 μm) grid serving as the size exclusion filter (6) by photolithography and dry etch processes. A 200-nm layer of gold was added to the size exclusion filter (6) by vapor deposition or coating of gold to serve as a gold electrode. A second layer of 300 μm thickness was made with silicon (110 or 200 μm wells) by the photolithography and dry etch processes to create a set of multiple microwells (18). The fabricated microwells layer was then mounted face up on the size exclusion filter (6), and the “top side” with the microwells (5) face up was further processed by etching electrode current lines (22) and filling with copper via electroplating and covering the lines with a protective layer to keep each of the microwells (5) readable.
The neutravidin was linked to the gold surface of the size exclusion filter (6) using the following functionalization procedure. The modification of the working electrode to functionalize the surface with neutravidin was performed by the 11-MUA, EDC, and HHSS method. This fabrication starts with dissolving 1.0 mM of 11-Mercapotundecanoic acid (11-MUA) into a 50 mM phosphate buffer solution at pH 10. Next, 150 μL of the solution is added to each well and allowed to sit overnight. The wells were washed with water 5 times and heated at 37° C. until dry. The terminal carboxylic groups (of 11-MUA) were then activated for 1 h by applying 150 μL of mM N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and 15 mM (N-hydroxy-succinimide ester (NHSS) in 50 mM phosphate buffer solution at pH 6.1. The sensor was washed with water 5 times and heated at 37 C until dry. Next, the surface of the working electrode was treated with 0.5 μL of neutravidin (Thermo fisher Prod. 31000) dissolved at 10.0 mg/mL into 50 mM phosphate buffer and reacted for 30 minutes to immobilize at 37° C. until dry. The sensor was washed with water 5 times and heated at 37 C until dry. In non-limiting examples, the neutravidin was replaced with alkaline phosphatase (1.7 mg/ml) and directly linked to the microwell.
After functionalization, the micro-filtration sensors were blocked with 200 μL solution of blocking buffer. The blocking buffer was made with 112.5 mL of water containing 10% Candor (Candor Bioscience, Cat. #110125), 3.18 g MOPSO, 1.50 g BSA (Bovine Serum Albumin (Fraction V), and 60 uL Proclin 200 and adjusted to pH 7.5 with 10 N sodium hydroxide and the buffer. After blocking overnight, the micro-filtration sensors were washed five times with 200 μL of TBS-T (Tris buffered saline with 0.05% Tween-20) and allowed to air dry.
The analyzer was built according to the schematic shown in
Mass reporters (17) were made of peptide with amino acids common to protein analytes tested are shown in Table 1. Mass reporters (17) were also made to synthetic homocysteine as a small molecule analyte and synthetic DNA oligo with a thiol modifier as a genetic analytes. These mass reporters (17) could be biotinylated (see example IC9-2B) and additionally contain a cleavable bond like the S_S (See example IC9-2B-S-S-RC-3-4 and IC9-2B-S-S-YC-9). Mass reporters (17) with biotin and cleavable bond were added manually in a buffer to specific microwells with vacuum and washed with water and vacuum until dry.
EC-IA and MS-IA analysis used biotinylated antibody (3) for capture of analytes (2), alkaline phosphate (ALP) or nanoparticle labeled antibody as second affinity agent (10), signal generating reagent (11) for electrochemical detection, and SIERRA nanoparticles as signal generating reagent (13) for mass spectrometric detection of mass labels (15). Reagents were added manually to buffer complex sample analyte and incubated at 37° C. until adding to the analyte detection microwell with microsensors and neutravidin linked to the surface of the size exclusion filter. The antibodies used in this example are specific for the analyte detected. In this example, polyclonal antibodies recognizing S. aureus (Thermo Fisher Scientific), E. coli (MyBioSource, San Diego, CA, USA), K. pneumoniae (Thermo Fisher Scientific) and P. aeruginosa (Abcam, Cambridge, UK) were used. The analytes for each sample were bacterial lysates prepared at 5×10{circumflex over ( )}3 to 5×10{circumflex over ( )}4 cell/mL as described (Pugia Anal Chem 2021). The antibody reagents used for a different analyte are kept separated in different microwells.
Liquids were moved by the analyzer by using the program control logic controller (PID) to automate the additions and vacuum. Step 1) was drawing complex sample with antibodies down into microwells through turning the vacuum on; Step 2) was keeping complex sample with antibodies in microwells for incubation through turning the vacuum off; Step 3) was incubation of antigen and antibodies complex in sensor microwells for 5 minutes to allow the antigen affinity complex to be captured by the neutravidin attached to a size exclusion filtration membrane through a linkage arm; and Step 4) was addition and removal of wash solutions five times as 200 μL of TBS-T (Tris buffered saline with 0.05% Tween-20) to allow removal of unbound materials. The analyzer accomplished these steps, using the peristaltic pump with linear actuator motors to dispense the TBS-T wash and the vacuum to remove the TBS-T wash from the size exclusion filtration membrane.
The electrochemical response of the EC-IA was measured by the analyzer by using the program control logic controller (PID) to record the reading of the potentiostat after addition of 100 μL of electrochemical solution containing para-amino-phenyl phosphate (pAPP) as the electrochemical reagent (13) which allowed generating para-amino phenol (pAP) as the electrochemical signal (12) by using ALP as the signal generating agent (11) or electrochemical solution of 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 100 mM Tris-HCl pH 8, and 0.2% Tween-20 by using the nanoparticle as the signal generating agent (11). The electrochemical signal generating is electrochemically measured with the potentiostat circuit board for calculating the response as a measurement of current changes vs voltage, as shown in
The analyzer used the potentiostat to read the sensor microwells and to measure and control voltages and current. The potentiostat circuit board allowed measurement of μAs across the working and reference/counter electrodes for −0.1 to 0.3 V. Each separate microwell was controlled through a multiplex board used in the potentiostat and the Arduino controller to deliver voltages and current results to the computer. The device was connected to a computer via a data storage card to provide all data from the Arduino for electrochemical analysis. The necessary hardware and electronics were fitted within a 12×21×6-inch case, including room for waste containment, three types of liquid reagents, the potentiostat, the microsensor and a small liquid crystal display (LCD) for the Arduino (PID).
For demonstration of capture and detection of bacteria, S. aureus, E. coli or P. aeruginosa were grown in culture, and commercial antibodies for said cells were used. Cell lysates were prepared by addition of BPEP-II surfactant. To make the antibody complex for capture, a sample contained 100 μL of the lysate sample (0, 5, 10, 20, 30, and 40 thousand cells or lysate equivalent per assay) added to 48 μL of the biotinylated S. aureus, E. coli, or P. aeruginosa polyclonal antibodies (0.75 μg/assay) and 30 μL of the same polyclonal antibodies conjugated to ALP (1.50 μg/assay), SIERRA reagent (100 μg/assay) or mass reporter label (1.50 μg/assay) and incubated for 1 hour at room temperature.
The potentiostat circuit board allowed measurements for the EC-IA analysis from 3 μA to 100 nA current across the working and reference/counter microelectrodes for −0.1 to 0.3 V. A 333 and 33 pM of ALP produced average current change of 2.4 and 0.8 μA at 0.2 V in 5 minutes using an electrochemical reaction solution with 1 mM pAPP, 100 mM TRIS (3.1 g/200 mL), 600 mM NaCl (7.0 g/200 mL), and 5 μM MgCl (0.2 g/200 mL) adjusted to pH 9.0. The ALP activity is optimal in basic pH range of 8 to 9, and falls rapidly to little reactivity at pH 6.3.
For MS-IA analysis, an additional 100 μg of the SIERRA MS-IA reagent was added. For mass reporter analysis, an additional 1.5 μg of IC9-2B-S-S-RC-3-4 or IC9-2B-S-S-YC-9 was added as mass reporters. After incubated at room temperature, along with polyclonal antibodies conjugated to ALP (1.50 μg/assay) for the electrochemical response of the EC-IA was measured as described above (Also see Pugia Anal Chem 2021). MS-IA mass labels (15) and mass reporters (17) were then collected after washing with acid solutions for breaking the C—O bonds or reducing agents for breaking the S-S.
Mass reporters (17) were made of synthetic homocysteine as a small molecule analyte and synthetic DNA oligo with a thiol modifier as a genetic analytes were released upon breakage of the —S—S— bond with reducing agents and were able to be detected by downstream analysis including optical methods, genetic methods such as polymerase chain reaction (PCR), molecular probes, immunoassays, and mass spectrometric (MS) detection technologies.
The concentration and spectra of released mass labels or mass reporters were determined using the LTQ-XL linear ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) fitted with a Dionex Ultimate 3000 autosampler as previous described (Pugia Anal Chem 2019, 2021). For MS analysis, the read signal for the assay used a different mass label from the read signal for the assay and the reporter signal used as the internal reference standard, which was used for calibration against a plot of peak intensity ratio of mass labels to mass reporters versus the concentration of analyte in solution. Representative mass spectra for a MS label or MS reporters were observed for each label.
For C—O nanoparticles, calibration solutions were made containing either AA-5, VV-5, IG-5 or GL-5 at 52.6, 26.3, 13.15, 6.58, 3.29, and 1.644 nM with 52.6 nM for VI-5 internal standards. All were prepared in 10 mM ammonium acetate pH 5.5 buffer with 1:1 methanol. For S-S nanoparticles, the calibration solutions contained AC-5 across the sample levels with 25 nM for AC-5.2 internal standard. Blank solutions were also prepared in the same buffer with 52.6 nM VI-5 or 25 nM for AC-5.2 internal standards. The RML signals in the calibration solutions and samples were measured by MS/MS in centroid mode. MS/MS scans were used to monitor unique fragments for RML and internal standards to determine the mass label concentrations and correlate them to the number of bacterial cells using calibration curves.
Surprisingly, the EC-IA analysis across −0.1 to 0.3 V did not break S-S bonds or cause acid formation to break C—O bonds. Application of a 0.2 V over 5 minutes using the electrochemical reactions solution with 333 and 33 pM ALP did not release any detectable mass labels for either C—O or S-S nanoparticles. For comparison, 0.001% citrate release buffer (pH 5.2) caused complete and immediate release of the mass labels (15) and mass reporters (17) utilizing —C—O— cleavable linkages, and 100 μL of 5 mM TCEP caused release of the mass labels (15) and mass reporters (17) utilizing —S—S— cleavable linkages over 30 minutes. mass labels (15) and mass reporters (17) utilizing —S—S— cleavable mass reporters are generated upon reduction at non-reducing voltages of <−0.1. The concentrations of MS-IA mass labels (15) and mass reporters (17) released under breakage condition did not suffer damage due the EC-IA analysis. The expected mass labels (15) and mass reporters (17) masses and concentrations were observed. While not bound to mode of actions, it is believed the rapid cycle times of the EC-IA analysis due to small microwells are fast enough to avoid reducing and acid-forming conditions. Optical fluorescent labels and mass labels could be added simultaneously with reagents capable of generating an electrochemical signal. Genetic analysis of cells by polymerase chain reaction (PCR) methods could be performed after generating an electrochemical response without interference. The polymerase chain reaction (PCR) method was also not interfered with by analysis using optical or mass spectrometric labels.
Mono-phosphate esters of substituted phenols, such para-amino-phenyl phosphate (pAPP), are highly reactant substrates for alkaline phosphatase (ALP). By way of example, multiple phosphate substrates have been studied including other aromatic rings and peptides but generally have no increased alkaline phosphatase (ALP) activity compared to substituted phenols, such as para-amino-phenol (pAP). The phenyl ring can be further substituted with a wide variety of organic atoms and groups along with any other group, such as fluorometric and optical labels, that do not increase alkaline phosphatase (ALP) activity.
Substituted phenols, such as para-amino-phenol (pAP) produced by ALP, are highly reactive, such that they undergo known auto-oxidation and dimerizing due to further oxidation by a second electron to reactive the para-quinone (pQ) species. These reactive pQ species are able to couple with aromatic phenols and amines in the Trinder's type reactions even under basic conditions (Pugia U.S. Pat. No. 5,362,633). These reactive pQ species are known to have potential to couple to a protein containing an attached phenolic residue, e.g., a tryrosine amino acid (Table 1).
To demonstrate a lack of interference for embodiment of the present disclosure, potential damage to peptides serving as examples of proteins as analytes for MS analysis were measured. The peptides contained Tryrosine (Tyr symbol) as a strong example of a receiving phenol for the para-quinone (pQ) (Table 1). After application of −0.1 to 0.2 V over 5 minutes using the electrochemical reactions solution with 333 and 33 pM ALP, no detectable changes to the mass labels were observed. A second voltage was applied at 0.1 V, and the pAP oxidization to para-quinone (pQ) occurred, as this is ideal voltage for the two-election oxidation with the loss of two electrons and two hydrogen from para-aminophenol (pAPP) to form para-quinone (pQ). Formation of this oxidation did not cause a coupling of the para-quinone (pQ) to the tryrosine of peptides in Table 1 or increase the mass or damage the mass label. Additionally, voltages greater than 0.1 V completely prevented the formation of para-quinone (pQ) to avoid any risk of damage to MS-IA mass labels (15) or mass reporters (17). The limit of how high this voltage needed to be was dependent on the electrode surface, working distance, and the composition of the electrochemical reaction solution; in this example, 0.2 V was sufficient to prevent damage.
A method demonstrating that electrochemical results of the electrochemical immunoassays (EC-IA) can be electronically coupled later to optical, genetic, or mass spectrometric results was performed using IC9-2B-S-S-RC-3-4 and IC9-2B-S-S-YC-9 as mass reporters. The electrochemical immunoassays (EC-IA) results were stored electrically, and electronic coupling to additional data required reading mass reporter mass and concentration at the expected values. The MS-IA reagents were added to the sample at the time of EC-IA but were processed after EC-IA analysis, which provided mass spectrometric results sequentially delayed. Mass reporters were measured by LTQ after all of the mass labels were collected from all of the microwells at the same time.
The IC9-2B-S-S-RC-3-4 and IC9-2B-S-S-YC-9 mass reporters produced unique mass spectrometric (MS) signals which are distinct from others and from those mass labels used to make a measurement of MS-IA. The mass reporter concentration was used as an indication of analyte integrity by comparison to acceptance limits. Comparison of observed concentration was within 90% of the expected concentration and indicated the analytes are valid for electronic linking to further analysis of analytes collected by optical, genetic, or mass spectrometric methods. The concentration and detection of mass reporter labels verify the analyte integrity as acceptable and valid for linking to EC-IA results to other associated electronic data (e.g., Sample ID, Patient ID, Sample Data, Patient Data, and the like).
The mass reporter labels (IC9-2B-S-S-RC-3-4 and IC9-2B-S-S-YC-9) were each placed into different wells of specifically defined positions in a sensor array where one well contained an analyte and the other well did not. The detection of the mass reporter labels was able to provide indication of specific microwells that held the mass reporter labels. This allowed identification of an analyte in microwells by the affinity agent added. EC-IA results indicated the presence of the analyte in a microwell and analysis of the mass reporter from that microwell indicated the analyte was suitable for linking to additional data for the analyte captured.
Additional mass reporters with unique masses and concentrations can be added to microwells and used for identification of analytes in a given microwell and to further demonstrate integrity or a lack of damage to an analyte by electrochemical reaction. The expected mass reporter signal and location is known at the time of manufacture and allows the diagnostic system to electronically compare measured mass reporter signals to expected mass reporters while using affinity agent location to identify the analyte and mass signal to verify analyte integrity. The analyte identity and integrity provided by mass reporters can be used for 1) reading additional standardization of mass spectrometric, optical, or PCR analysis; and 2) automatic correction of integrity of an analyte for stability as well other associated factors impacting integrity.
In all examples, the reagents for electrochemical, optical, and mass spectrometric detection could be processed together in one common electrochemical sensor microwell with a size exclusion filter, electrodes, and affinity agents for analyte reagent capture and analyte detection. In all examples, affinity agents allowed the capture of cells, biomolecule analytes, and mass reporters.
In some examples, the affinity agents for analyte capture also allow the capture of a mass reporter used to determine the identity and integrity of the analyte and sample being suitable for additional linking data. In other examples, a mass reporter is used as a marker of microwell location for an analyte released for detection by additional optical, genetic, and/or mass spectrometric analysis. In other examples, a mass reporter is used as an internal standard for an analyte released for detection by additional optical, genetic, and/or mass spectrometric analysis.
While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the representative embodiments have been shown and described and that all changes, equivalents, and modifications that come within the spirit of the inventions defined by the claims are desired to be protected. All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference and set forth in its entirety herein.
This application is the United States national phase of International Application No. PCT/US21/55402 filed Oct. 18, 2021, and claims priority to U.S. Provisional Patent Application No. 63/092,860, filed Oct. 16, 2020, the disclosures of which are hereby incorporated by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/55402 | 10/18/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63092860 | Oct 2020 | US |
