Immobilized protein system for rapid and enhanced multiplexed diagnostics

Abstract

The present invention relates to methods of detecting a neural injury biomarker in a biological sample. The method includes subjecting a biological sample to an assay according to the present invention that produces a measurable signal and detecting the measurable signal. The presence or absence of the measurable signal indicates the presence or absence of the biomarker in the sample. The present invention also relates to methods of determining the state of a subject's neural injury. The present invention also relates to systems and devices useful in carrying out the methods of the present invention.

1 . A method for detecting the presence or absence of a neural injury biomarker in a biological sample, the method comprising:
 providing a biological sample;   subjecting the biological sample to an assay to detect the neural injury biomarker selected from the group consisting of:
 (a) an assay to detect S100β, wherein the assay comprises (i) providing aldolase, glyceraldehyde 3-phosphate dehydrogenase (“GAPDH”), and a signal-transducing molecule, each immobilized on one or more supports, (ii) providing fructose 1,6-bisphosphate and NAD + , and (iii) contacting the biological sample with the one or more supports, NAD + , and fructose 1,6-bisphosphate under conditions effective to cause a cascading biological reaction leading to production of a measurable signal by the signal-transducing molecule if S100β is present in the biological sample; 
 (b) an assay to detect S100β, wherein the assay comprises (i) providing phosphoglyceromutase (“PGM”), enolase, pyruvate kinase, and a signal-transducing molecule, each immobilized on one or more supports, (ii) providing 3-phosphoglycerate and (iii) contacting the biological sample with the one or more supports and 3-phosphoglycerate under conditions effective to cause a cascading biological reaction leading to production of a measurable signal by the signal-transducing molecule if S100β is present in the biological sample; 
 (c) an assay to detect glial fibrillary acidic protein (“GFAP”), wherein the assay comprises (i) providing a GFAP-phosphorylating kinase and luciferase, each immobilized on one or more supports, (ii) providing D-luciferin, O 2 , and ATP, and (iii) contacting the biological sample with the one or more supports, D-luciferin, O 2 , and ATP under conditions effective to permit a sequential reaction, whereby GFAP, if present in the biological sample, will react with the GFAP-phosphorylating kinase and ATP thereby producing ADP, whereby a decreased amount of ATP reacts with luciferase to produce a measurable signal compared to when GFAP is not present in the biological sample and the sequential reaction does not take place; 
 (d) an assay to detect a neural injury biomarker, wherein the neural injury biomarker is an enzyme, the assay comprising (i) providing one or more substrates of the neural injury-biomarker and one or more co-factors, (ii) providing one or more enzymes capable of acting upon the product of a reaction between the neural injury biomarker and the one or more substrates, wherein at least one enzyme is immobilized on one or more supports and at least one of the one or more enzymes is a signal-transducing molecule; and (iii) contacting the biological sample with the one or more substrates and the one or more enzymes under conditions effective to permit a sequential reaction, whereby the neural injury biomarker, if present in the biological sample, will react with the one or more substrates causing a single reaction or a series of coupled reactions that cause production of a measurable signal by the signal-transducing molecule; 
 (e) combinations of two or more of (a), (b), (c), and (d); and 
   detecting, based on said subjecting, the measurable signal, wherein the measurable signal indicates presence or absence of the neural injury biomarker in the biological sample.   
     
     
         2 . The method according to  claim 1 , wherein the signal-transducing molecule of the assay of (a), (b), and/or (d) is selected from the group consisting of firefly luciferase, renilla luciferase, horseradish peroxidase, pyruvate oxidase, and a chromogen. 
     
     
         3 . The method according to  claim 1 , wherein the measurable signal of the assay of (a), (b), (c), and/or (d) is a photometrically detectable signal or a colorimetrically detectable signal. 
     
     
         4 . The method according to  claim 1 , wherein the biological sample is subjected to the assay of (a) and wherein the aldolase, GAPDH, and signal-transducing molecule are each immobilized on separate supports. 
     
     
         5 . The method according to  claim 1 , wherein the biological sample is subjected to the assay of (a), the signal-transducing molecule is the chromogen tetrazolium chloride, and the cascading biological reaction comprises S100β modulating the activity of aldolase to increase the fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate conversion rate, the glyceraldehyde 3-phosphate reacting with GAPDH and NAD + to produce 1, 3-bisphosphoglycerate and NADH with the NADH reacting with the chromogen tetrazolium chloride to produce a measurable signal. 
     
     
         6 . The method according to  claim 1 , wherein the biological sample is subjected to the assay of (a) and luciferase is the signal-transducing molecule, said assay further comprising:
 providing phosphoglycerate kinase (“PGK”), ADP, D-luciferin, and O 2 , wherein the PGK, ADP, D-luciferin, and O 2  are contacted with the biological sample during said contacting, said cascading biological reaction comprising S100β modulating the activity of aldolase to increase the fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate conversion rate, the glyceraldehyde 3-phosphate reacting with GAPDH and NAD +  to produce 1, 3-bisphosphoglycerate and NADH with the 1, 3-bisphosphoglycerate and ADP reacting with PGK to produce 3-phosphoglycerate and ATP, said ATP reacting with luciferase to produce a measurable signal.   
     
     
         7 . The method according to  claim 1 , wherein the biological sample is subjected to the assay of (b) and wherein the PGM, enolase, pyruvate kinase, and signal-transducing molecule are each immobilized on separate supports. 
     
     
         8 . The method according to  claim 1 , wherein the biological sample is subjected to the assay of (b) and the signal-transducing molecule is luciferase, said assay further comprising:
 providing D-luciferin and O 2 , wherein the D-luciferin and O 2  are contacted with the biological sample during said contacting, said cascading biological reaction comprising S100β modulating the activity of PGM to increase the 3-phosphoglycerate to 2-phosphoglycerate conversion rate, the 2-phosphoglycerate reacting with enolase to produce phosphoenolpyruvate (“PEP”) and ADP with the PEP and ADP reacting with pyruvate kinase to produce pyruvate and ATP, said ATP reacting with luciferase to produce a measurable signal.   
     
     
         9 . The method according to  claim 1 , wherein the biological sample is subjected to the assay of (c) and the GFAP-phosphorylating kinase and luciferase are each immobilized on separate supports. 
     
     
         10 . The method according to  claim 1 , wherein the biological sample is subjected to the assay of (c) and the GFAP-phosphorylating kinase is selected from the group consisting of rho-associated protein kinase (“ROCK”), ROCK1, ROCK2, and Aurora-B kinase. 
     
     
         11 . The method according to  claim 1 , wherein the biological sample is subjected to the assay of (c), wherein the assay further comprises:
 providing a GFAP-dephosphorylating phosphatase and contacting the biological sample with the GFAP-dephosphorylating phosphatase prior to said contacting of (iii).   
     
     
         12 - 15 . (canceled) 
     
     
         16 . The method according to  claim 1 , wherein the biological sample is subjected to the assay of (a), (b), and/or (d) and the presence of the measurable signal indicates the presence of the neural injury biomarker. 
     
     
         17 . The method according to  claim 1 , wherein the biological sample is subjected to the assay of (c) and wherein reduction in the measurable signal, compared to when GFAP is not present in the biological sample, indicates presence of the GFAP in the biological sample. 
     
     
         18 . (canceled) 
     
     
         19 . The method according to  claim 1 , wherein the support(s) comprise(s) a nanoparticle. 
     
     
         20 . (canceled) 
     
     
         21 . The method according to  claim 1  further comprising:
 providing a substrate comprising one or more channels and subjecting the biological sample to the assay within the one or more channels. 
 
     
     
         22 . (canceled) 
     
     
         23 . The method according to  claim 1  further comprising:
 subjecting the biological sample to at least a second assay different than the assays of (a), (b), (c), and (d) to detect a neural injury biomarker, wherein the second assay produces a measurable signal if the neural injury biomarker is present in the sample and 
 detecting, based on said subjecting the biological sample to the at least second assay, the measurable signal produced by the second assay, wherein the measurable signal produced by the second assay indicates presence or absence of the neural injury biomarker in the biological sample. 
 
     
     
         24 - 46 . (Cancelled) 
     
     
         47 . A system comprising:
 a platform comprising one or more channels, at least one channel of said one or more channels comprising:   (a) biological assay components suitable to detect the presence of S100β in a biological sample, said components comprising: (i) aldolase, glyceraldehyde 3-phosphate dehydrogenase (“GAPDH”), and a signal-transducing molecule, each immobilized within the at least one channel and (ii) fructose 1,6-bisphosphate and NAD + , said components positioned in the channel to permit a cascading biological reaction leading to production of a measurable signal by the signal-transducing molecule if S100β is present in the sample;   (b) biological assay components suitable to detect the presence of S100β in a biological sample, said components comprising: (i) phosphoglyceromutase (“PGM”), enolase, pyruvate kinase, and a signal-transducing molecule, each immobilized within the at least one channel and (ii) 3-phosphoglycerate, said components positioned in the channel to permit a cascading biological reaction leading to production of a measurable signal by the signal-transducing molecule if S100β is present in the sample;   (c) biological assay components suitable to detect the presence of GFAP in a biological sample, said components comprising: (i) a GFAP-phosphorylating kinase and luciferase, each immobilized within the at least one channel, (ii) D-luciferin, O 2 , and ATP, said components positioned in the at least one channel to permit a sequential reaction in the presence of GFAP, whereby GFAP will react with the GFAP-phosphorylating kinase and ATP to produce ADP;   (d) biological assay components suitable to detect the presence of a neural injury biomarker in a biological sample, wherein the neural injury biomarker is an enzyme, said components comprising: (i) one or more substrates of the neural injury biomarker and one or more co-factors; (ii) one or more enzymes capable of acting upon the product of a reaction between the neural injury biomarker and the one or more substrates, wherein at least one enzyme is immobilized within the at least one channel on one or more supports and at least one enzyme is a signal-transducing molecule, said components positioned in the channel to permit a sequential reaction, whereby the neural injury biomarker, if present in the biological sample, will react with the one or more substrates causing a single reaction or a series of coupled reactions that cause production of a measurable signal by the signal-transducing molecule; or   (e) combinations of two or more of (a), (b), (c), and (d).   
     
     
         48 . The system according to  claim 47  further comprising:
 at least one of the one or more channels that serves as a negative control. 
 
     
     
         49 . The system according to  claim 47  further comprising:
 at least one of the one or more channels that serves as a positive control. 
 
     
     
         50 . The system according to  claim 47  further comprising:
 a measurable signal detector. 
 
     
     
         51 . (canceled)