Detection of bioagents using a shear horizontal surface acoustic wave biosensor

Abstract

Viruses and other bioagents are of high medical and biodefense concern and their detection at concentrations well below the threshold necessary to cause health hazards continues to be a challenge with respect to sensitivity, specificity, and selectivity. Ideally, assays for accurate and real time detection of viral agents and other bioagents would not necessitate any pre-processing of the analyte, which would make them applicable for example to bodily fluids (blood, sputum) and man-made as well as naturally occurring bodies of water (pools, rivers). We describe herein a robust biosensor that combines the sensitivity of surface acoustic waves (SAW) generated at a frequency of 325 MHz with the specificity provided by antibodies and other ligands for the detection of viral agents. In preferred embodiments, a lithium tantalate based SAW transducer with silicon dioxide waveguide sensor platform featuring three test and one reference delay lines was used to adsorb antibodies directed against Coxsackie virus B4 or the negative-stranded category A bioagent Sin Nombre virus (SNV), a member of the genus Hantavirus, family Bunyaviridae, negative-stranded RNA viruses. Rapid detection (within seconds) of increasing concentrations of viral particles was linear over a range of order of magnitude for both viruses, although the sensor was approximately 50×10 4 -fold more sensitive for the detection of SNV. For both pathogens, the sensor's selectivity for its target was not compromised by the presence of confounding Herpes Simplex virus type 1. The biosensor was able to detect SNV at doses lower than the load of virus typically found in a human patient suffering from hantavirus cardiopulmonary syndrome (HCPS). Further, in a proof-of-principle real world application, the SAW biosensor was capable of selectively detecting SNV agents in complex solutions, such as naturally occurring bodies of water (river, sewage effluent) without analyte pre-processing.

1 - 52 . (canceled) 
     
     
         53 . A ligand based biosensor comprising a biological ligand complexed, directly or indirectly to a surface of a piezoelectric material, said piezoelectric material being operably connected to an electric circuit capable of producing surface acoustic waves on the surface of the piezoelectric material to detect the binding of a bioagent or analyte to said ligand based biosensor, wherein said bioagent or analyte is selected from the group consisting of viruses and their extracts, prions and their extracts, eucaryotic cells, fragments and extracts, prokaryotic cells, fragments and extracts, prokaryotic spores, fragments and extracts, protein and peptide markers, serum proteins, isolated proteins, polypeptides, synthethic chemicals, viral membranes, eukaryotic membranes, eukaryotic cell parts, including mitochondria and other organelles, prokaryotic membranes, DNA viruses (single and double stranded DNA viruses), RNA viruses (single and double stranded RNA viruses), point mutations (any organism), single nucleotide polymorphism (any organism), mRNA's, rTNAs or a micro RNA. 
     
     
         54 . The biosensor according to  claim 53  wherein said biological ligand is a polypeptide, a DNA or RNA molecule, a lipid or carbohydrate. 
     
     
         55 . The biosensor according to  claim 53  wherein said piezoelectric material is lithium tantalate ((LiTaO 3 ). 
     
     
         56 . The biosensor according to  claim 53  wherein said ligand is an antibody. 
     
     
         57 . The biosensor according to  claim 56  wherein said antibody is a monoclonal antibody. 
     
     
         58 . The biosensor according to  claim 56  wherein said antibody is a polyclonal antibody. 
     
     
         59 . The biosensor according to  claim 53  wherein said ligand is a polypeptide. 
     
     
         60 . The biosensor according to  claim 53  wherein said ligand is polynucleotide. 
     
     
         61 . The biosensor according to  claim 60  wherein said ligand is a DNA molecule. 
     
     
         62 . The biosensor according to  claim 60  wherein said ligand is a RNA molecule. 
     
     
         63 . The biosensor according to  claim 53  wherein said ligand is a lipid or a carbohydrate. 
     
     
         64 . The biosensor according to  claim 53  wherein said ligand is covalently linked to said piezoelectric material. 
     
     
         65 . The biosensor according to  claim 53  wherein said ligand is non-covalently linked to said piezoelectric material. 
     
     
         66 . The biosensor according to any of  claim 53  wherein said bioagent or analyte is selected from the group consisting of viruses and their extracts, prions and their extracts, eucaryotic cells, fragments and extracts, prokaryotic cells, fragments and extracts, prokaryotic spores, fragments and extracts, protein and peptide markers, serum proteins, isolated proteins, polypeptides, synthethic chemicals, viral membranes, eukaryotic membranes, eukaryotic cell parts, including mitochondria and other organelles, prokaryotic membranes, DNA viruses (single and double stranded DNA viruses), RNA viruses (single and double stranded RNA viruses), point mutations (any organism), single nucleotide polymorphism (any organism), mRNA's, rTNAs or a micro RNA. 
     
     
         67 . The biosensor according to  claim 53  wherein said bioagent is a protein marker or polypeptide. 
     
     
         68 . The biosensor according to  claim 67  wherein said bioagent is a peptide marker or polypeptide. 
     
     
         69 . A method of identifying the presence of a bioagent in a sample with the ligand based biosensor according to  claim 53 , said method comprising establishing a surface acoustic wave on the surface of said piezoelectric material comprising a ligand which binds to said bioagent, exposing said surface to a sample suspected of containing said bioagent and then determining whether the sample contains a suspect bioactive agent if the wave at the surface of said piezoelectric material evidences a change consisting with the binding of said bioactive agent. 
     
     
         70 . The method according to  claim 69  wherein said sample is an environmental or human sample. 
     
     
         71 . The method according to  claim 70  wherein said human sample is blood, serum, plasma, urine, sputum or fecal matter. 
     
     
         72 . The method according to  claim 69  wherein said surface acoustic wave has a specific resonance frequency ranging from 80 to 400 Mhz. 
     
     
         73 . The method according to  claim 69  wherein said surface acoustic wave has a specific resonance frequency of about 325 Mhz. 
     
     
         74 . The method according to  claim 69  wherein said bioagent or analyte is selected from the group consisting of viruses and their extracts, prions and their extracts, eucaryotic cells, fragments and extracts, prokaryotic cells, fragments and extracts, prokaryotic spores, fragments and extracts, protein and peptide markers, serum proteins, isolated proteins, polypeptides, synthethic chemicals, viral membranes, eukaryotic membranes, eukaryotic cell parts, including mitochondria and other organelles, prokaryotic membranes, DNA viruses (single and double stranded DNA viruses), RNA viruses (single and double stranded RNA viruses), point mutations (any organism), single nucleotide polymorphism (any organism), mRNA's, rTNAs or a micro RNA. 
     
     
         75 . A ligand based biosensor comprising a lithium tantalate material and at least one biological ligand complexed, directly or indirectly to the surface of said material, said material further comprising at least two delay lines wherein at least one of said delay lines is a reference delay line which produces a surface acoustic wave at the surface of the biosensor and at least one of said delay lines is a test delay line which measures the frequency of the acoustic wave at the surface of the biosensor to detect the binding of a bioagent or analyte to said ligand.