Sensors and methods for detecting diseases caused by a single point mutation

A method of determining a presence and quantity of Factor V Leiden (FVL) and Normal Factor V (FVN) in a sample, the method comprising:

• providing a first monoclonal antibody and a second monoclonal antibody,
• wherein the first monoclonal antibody was obtained against a peptide comprising FVN-20mer (SEQ ID NO 1):
• H-I-C-K-S-R-S-L-D-R-R-G-I-Q-R-A-A-D-I-E-Q-NH2 and specifically binds to native FVN as found in human plasma, wherein binding affinity of the first antibody to native FVN is greater than the binding affinity of the first antibody to native FVL as found in human plasma of a person with FVL abnormality;
• wherein the second monoclonal antibody was obtained against a peptide comprising FVL-20-mer (SEQ ID NO:
• 2):
• H-I-C-K-S-R-S-L-D-R-Q-G-I-Q-R-A-A-D-I-E-Q-NH2 and specifically binds to native FVL as found in human plasma of a person with FVL abnormality and wherein the binding affinity of the second antibody to native FVL is greater than the binding affinity of the second antibody to native FVN;
• immobilizing the first antibody and the second antibody on first and second optical sensing fibers, respectively;
• exposing the sample to each of the immobilized antibodies separately to bind FVN and/or FVL in the sample to the optical sensing fibers;
• probing the first and second sample-exposed optical sensing fibers with a fluorophore-tagged antibody that binds to both bound FVN and FVL with equal affinity;
• obtaining fluorescent signal intensity values from bound fluorophore-tagged antibody for each of the first optical sensing fiber and the second optical sensing fiber;
• and
• solving a system of equations with the obtained signal intensity values to determine unknown values associated with respective concentrations of FVN and FVL in the sample, wherein the presence and respective quantities of FVN and FVL are determined according to the following system of equations:
• SITOTAL for FVN preferable sensor (SI1)=A1×CFVN+B1×CFVL
• SITOTAL for FVL preferable sensor (SI2)=A2×CFVN+B2×CFVL
• where SI1 represents signal intensity for the FVN preferable first optical fiber sensor, SI2 represents signal intensity for the FVL preferable second optical fiber sensor, A1 represents a slope of a standard curve showing a relationship between concentration and signal intensity of the FVN preferable sensor for FVN using samples with FVN only, A2 represents a slope of a standard curve showing a relationship between concentration and signal intensity of the FVL preferable sensor for FVN using samples with FVN only, B1 represents a slope of a standard curve showing a relationship between concentration and signal intensity for FVL using the samples with FVL only for the FVN preferable sensor, B2 represents a slope of a standard curve showing a relationship between concentration and signal intensity for FVL using the samples with FVL only for the FVL preferable sensor, and C represents concentration to be determined.

An immunological sensor for determining a presence and quantity of Factor V Leiden (FVL) and Normal Factor V (FVN) in a sample comprising:

• a housing having a microfluidic network disposed therein;
• first and second optical sensing fibers disposed within said microfluidic network and configured to be in fluid communication with one another, said first optical sensing fiber having a first monoclonal antibody immobilized thereon and a second optical sensing fiber having a second monoclonal antibody immobilized thereon,
• wherein the first monoclonal antibody was obtained against a peptide comprising FVN-20mer (SEQ ID NO 1):
• H-I-C-K-S-R-S-L-D-R-R-G-I-Q-R-A-A-D-I-E-Q-NH2 and specifically binds to native FVN as found in human plasma and wherein the binding affinity of the first antibody to native FVN is greater than the binding affinity of the first antibody to native FVL as found in human plasma of a person with FVL abnormality;
• wherein the second monoclonal antibody was obtained against a peptide comprising FVL-20-mer (SEQ ID NO:
• 2):
• H-I-C-K-S-R-S-L-D-R-Q-G-I-Q-R-A-A-D-I-E-Q-NH2 and specifically binds to native FVL as found in human plasma of a person with FVL abnormality wherein the binding affinity of the second antibody to native FVL is greater than the binding affinity of the second antibody to native FVN;
• an inlet for sample application to the microfluidic network and an outlet for discharging the sample following analysis, said inlet and said outlet configured to be in fluid communication with both said first and second optical sensing fibers and with one another;
• and
• a signal detector for detecting respective fluorescence signal emissions of said first and second optical sensing fibers following exposure of said first and second optical sensing fibers first to the sample and subsequently to a fluorophore-tagged antibody possessing equal affinity to both FVN and FVL when bound to the optical sensing fibers.