Method for analysis of aerosolized biological species in epidemic and pandemic prediction

Abstract

The disclosed invention is a new concept for a network of specialized continuous ambient air sampling systems, which employ a novel non-destructive ionization and separation method, coupled to a near real-time genomic sequencer. The network of preferably pathogen samplers, would constitute a plurality of distributed nodes throughout the world, with bacterial & viral concentration, identification, and mutation data uploaded to the cloud for epidemic/pandemic predictive modeling. The proposed system offers the ability to migrate from outbreak surveillance, to outbreak forecast. In addition, the capability for continuous data of genomic sequencing offers an enhanced capability to help track antigenic drift and antigenic shift. While optimized for viral capture and analysis, any airborne pathogen or spore can be accepted using the technology. Applications include world health monitoring, pandemic prediction, and detection of real-time bioterror pathogen deployment.

Claims

1. A device to surveil and forecast public health and force protection pathogen threats, including viral outbreaks, the device comprising: (a) an air sampler utilizing aerosol impaction filtration, causing heavier fractions to impact and embed in an end channel, lighter fractions negotiating the change in direction, subsequently lighter fractions can be derived through a stratification based on mass through an airflow; (b) a cross current or counter current electrospray source employed to nondestructively capture bioaerosols from the air flow, and remove them from the air flow via electrostatic forces; (c) filtration accomplished using an electrostatic filter, preliminary identification and separation of a virion or a biospecies achieved using a charge detection mass spectrometer; (d) bioaerosols accumulated on a witness plate, in preparation for automated sequencing in a sequencer, the sequencer being a nanopore device, and the sequencer outputting a genetic sequence; (e) electrospray charged pathogens sampled away from an aerosol pre-filter, an image charge produced each time a pathogen particle passes through a flight tube, a viral or bacterial count obtained in addition to m/z charge spectral information about a specific pathogen or virion selected; (f) an ionic liquid acting as an electrolyte in nanopore sequencer, or a silicone based diffusion pump oil seeded with an ionic liquid; and wherein the device is configured to perform a low pressure viral lysis via ionic liquids.

2. The device of claim 1, wherein the charge detection mass spectrometer comprises: desorbing electrospray droplets to which ambient polar or polarizable biological trace species in the air are attracted, and absorbed by the droplets, resulting in discrete charged particles, a capillary tube configured to receive and pass through the charged biological particles into a succession of progressively pumped regions separated by skimmers and through an image charge detector tube; a picoammeter and computer configured to detect and amplify the image charge of charged biological trace species; a target that consists of a nanopore sequencer, the target configured to both lyse biological trace species and allow a charge fluctuation of nucleic acid or other biological molecular group to pass through the nanopore detector thereby identifying each molecular or atomic group or species thereof.

3. The device of claim 2, wherein the trace species are electrospray charged bioaerosol trace species, devoid of solvent or aqueous solution, and introduced into a preferably partial pressure region, passing through a charge detection mass spectrometer tube, electrostatically focused, deflected by horizontal and vertical plates, scanned in a raster pattern or simply directed to a specific target on a nanopore detection surface, said charged bioaerosol trace species consisting of particulates or virions or nucleic acid or bacteria, or protein or peptide or any bio-species.

4. The device of claim 3, wherein the charge detection mass spectrometer tube, and horizontal and vertical plates, are configured to scan the scanned in raster pattern in a successive raster pattern with a beginning point and a terminal point, before the raster pattern is retraced to the next level below or down next to and parallel to a preceding row of scan targets, each target represents an individual ionic liquid nanopore well, and successive wells permit selective analysis depending upon the composition of the electrospray charged bioaerosol trace species.

5. The device of claim 2, further comprising: conductive fluid driven by either capillary action or hydrostatic force into an electrically conductive needle, where a difference of electric potential is applied between the needle and the target using a power supply configured to create a Taylor Cone from which emerges a jet of fluid which as the fluid evaporates, results in desorbing droplets, said droplets carrying a surface charge such that ambient biological species are attracted and absorbed by aforementioned desorbing droplets, until only charged biological trace species remain and are deposited onto an opposing electrical pole target, which is a nanopore with an electrolyte on either face, said electrolyte preferably being a low volatility ionic liquid that will not evaporate, to which a difference of electrical potential has been applied.

6. The device of claim 1, further comprising a low vapor pressure grease located in the air sampler utilizing aerosol impaction filtration and configured to allow heavy fraction to be permanently entrained.

7. The device of claim 1, wherein the charge detection mass spectrometer is configured to operate at or near atmospheric pressure.

8. The device of claim 1 further comprising: an electrostatic gating system where arriving pathogen particles can be alternately introduced into the genetic sequencer, or discharged and collected for subsequent study, or destroyed as desired.

9. The device of claim 1, further comprising: nanopore sequencer cells, arranged in a matrix such that each cell can be selected for new genome sample processing depending on the desired type of virion being interrogated, and wherein the charged virions emitted from the charge detection mass spectrometer and the charged virions are electrostatically or magnetically deflected to a desired sequencing cell.

10. The device of claim 1, wherein the sequencer is coated with a non volatile electrolyte.

11. The device of claim 10, wherein the non volatile electrolyte is ionic liquid.

12. The device of claim 1, wherein the sequencer is configured to upload the genetic sequence to a cloud computing network.

13. The device of claim 1, wherein the sequencer is configured to perform a comparative analysis of the genetic sequence a genetic sequence stored in a database.

14. A system to surveil and forecast public health and force protection pathogen threats, including viral outbreaks, the system comprising: a first device comprising: (a) an air sampler utilizing aerosol impaction filtration, causing heavier fractions to impact and embed in an end channel, lighter fractions negotiating the change in direction, subsequently lighter fractions can be derived through a stratification based on mass through an airflow; (b) a cross current or counter current electrospray source employed to nondestructively capture bioaerosols from the air flow, and remove them from the air flow via electrostatic forces; (c) filtration accomplished using an electrostatic filter, preliminary identification and separation of the virion or biospecies achieved using an ion mobility spectrometer; (d) bioaerosols accumulated on a witness plate, in preparation for automated sequencing in a sequencer, the sequencer being a nanopore device, and the sequencer outputting a genetic sequence; (e) electrospray charged pathogens sampled away from an aerosol pre-filter, an image charge produced each time a pathogen particle passes through a flight tube, a viral or bacterial count obtained in addition to m/z charge spectral information about a specific pathogen or virion selected; (f) an ionic liquid configured as an electrolyte in nanopore sequencer, or a silicone based diffusion pump oil seeded with an ionic liquid; (g) low pressure viral lysis via the ionic liquid; a second device located away from the first device, the second device comprising: (a) an air sampler utilizing aerosol impaction filtration, causing heavier fractions to impact and embed in an end channel, lighter fractions negotiating the change in direction, subsequently lighter fractions can be derived through a stratification based on mass through an airflow; (b) a cross current or counter current electrospray source employed to nondestructively capture bioaerosols from the air flow, and remove them from the air flow via electrostatic forces; (c) filtration accomplished using an electrostatic filter, preliminary identification and separation of the virion or biospecies achieved using an ion mobility spectrometer; (d) bioaerosols accumulated on a witness plate, in preparation for automated sequencing in a sequencer, the sequencer being a nanopore device, and the sequencer outputting a genetic sequence; (e) electrospray charged pathogens sampled away from an aerosol pre-filter, an image charge produced each time a pathogen particle passes through a flight tube, a viral or bacterial count obtained in addition to m/z charge spectral information about a specific pathogen or virion selected (f) an ionic liquid configured as an electrolyte in nanopore sequencer, or a silicone based diffusion pump oil seeded with an ionic liquid; and (g) low pressure viral lysis via the ionic liquid; a third device located away from the first device and second device, the third device comprising: (a) an air sampler utilizing aerosol impaction filtration, causing heavier fractions to impact and embed in an end channel, lighter fractions negotiating the change in direction, subsequently lighter fractions can be derived through a stratification based on mass through an airflow; (b) a cross current or counter current electrospray source employed to nondestructively capture bioaerosols from the air flow, and remove them from the air flow via electrostatic forces; (c) filtration accomplished using an electrostatic filter, preliminary identification and separation of the virion or biospecies achieved using an ion mobility spectrometer; (d) bioaerosols accumulated on a witness plate, in preparation for automated sequencing in a sequencer, the sequencer being a nanopore device, and the sequencer outputting a genetic sequence; (e) electrospray charged pathogens sampled away from an aerosol pre-filter, an image charge produced each time a pathogen particle passes through a flight tube, a viral or bacterial count obtained in addition to m/z charge spectral information about a specific pathogen or virion selected; (f) an ionic liquid configured as an electrolyte in nanopore sequencer, or a silicone based diffusion pump oil seeded with an ionic liquid; and (g) low pressure viral lysis via the ionic liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Charge Detection & Filtration of Specific Virions:

(2) Electrospray has been demonstrated to non-destructively add charge to biological species, such as spores, bacteria, and viruses. Viruses that have been charged using electrospray have been shown to be capable of being coarsely separated from an air steam. So what do we do next to identify viruses that have been separated in an airstream? A way is needed to further filter virion particles, and concentrate them onto a surface for subsequent genetic sequencing.

(3) For over three decades since John Fenn's discovery at Yale, electrospray ionization (ESI) has been used to ionize non-covalent complexes and subsequently transfer the intact ion into the gas phase for mass spectrometry (MS) analysis. ESI generates a distribution of multiple charged ions, resulting in a m/z spectrum comprised of a series of peaks, known as a charge state envelope. To obtain mass information, the number of charges for each peak must be deduced. For smaller biological analytes like peptides, the charge states are sufficiently resolved and this process is straightforward. For macromolecular complexes exceeding ˜100 kDa, this process is complicated by the broadening and shifting of charge states due to incomplete desolvation, salt adduction, and inherent mass heterogeneity. As the analyte mass approaches the MDa regime, the m/z spectrum is often comprised of a broad distribution of unresolved charge states. In such cases, mass determination is precluded. Charge Detection Mass Spectrometry (CDMS) is an emerging MS technique for determining the masses of heterogeneous, macromolecular complexes. In CDMS, the m/z and z of single ions are measured concurrently so that mass is easily calculated. With this approach, deconvolution of a m/z spectrum is unnecessary. This measurement is carried out by passing macroions through a conductive cylinder. The induced image charge on the cylindrical detector provides information about m/z and z: the m/z is related to its time-of-flight through the detector, and the z is related to the intensity of the image charge. As a result, given the megadalton masses of many viruses (Influenza average value was 174×10.sup.6 daltons), the application of charge detection spectrometric techniques is appropriate to the present tasking.

(4) As electrospray charged virions are sampled away from the aerosol pre-filter, an image charge will be produced each time a virus particle passes through the flight tube, FIG. 3. Therefore, a viral count can be obtained (analogous to blood cell counts in a Coulter counter), in addition to m/z charge spectral information about the specific virion selected. Knowing the mass and the viral counts, the quantity of viral particles can be deduced as collected on the witness plate or analysis target inside the CDMS system. Deposition on the discharge or witness plate is the last step before introduction to the genetic sequencer, if the target is not the sequencer itself. Being charged species, an electrostatic gating system may be created where arriving viral particles can be alternately introduced into the genetic sequencer front end, or discharged and collected for subsequent study, or destroyed as desired.

(5) It should be noted that inventive claims extend to charge detection spectometry performed at or near atmospheric pressure. In addition, ion mobility may be substituted for charge detection spectrometry at or near atmospheric pressure for charged bio-species sorting subsequent to preferred nanopore identification.

(6) Viral Lysis

(7) Several lysis methods may be employed in the sampler. Traditional reagent-thermal based methods may be employed, or, a reagentless electrical lysis approach may be employed, which has been demonstrated for vaccinia virions. Using a pair of nano electrodes, an externally applied electric field produces the trans-membrane potential, and it generates electric field inside the lipid bilayer membrane. This internal electric field generates a Maxwell stress, compressing the membrane in the normal direction, and rupture the membrane if the electric field is above critical value. Electric lysis is virtually instantaneous, whereas traditional reagent based approaches require up to 30-45 minutes per sample.

(8) One aspect of standard reagent lysis is the fact that the process is performed using aqueous fluids at atmospheric pressure. In ESI-MS and ESI-CDMS, the collected species will be subjected to a partial pressure region, where aqueous fluids would instantaneously evaporate. One solution to low pressure viral lysis, aside from the aforementioned electric field method, is the use of ionic liquids, which have recently been shown to not only lyse virion capsids, but do so in some cases at higher efficiency than commercial reagent kits. Ionic liquids are room temperature salts, which have essentially zero vapor pressure. In addition, ionic liquids may serve an added purpose in helping perform genetic sequencing, discussed in the section on improved nanopore sequencing.

(9) Sequencing

(10) Virions in the airstream, once ionized by electrospray, filtered and sequestered based on mass and charge information, are now ready for sequencing. As indicated earlier, the preferred sequencing device is a graphene nanopore system, although any sequencer could in theory be utilized. A graphene nanopore device deduces genetic sequences based on an ionic current through a set of nano sized voids or pores, and measures the changes in current as biological molecules pass through the pore or near it. The information about the change in current can be used to identify that molecule compared to a library. A nanopore can be thought of as a genetic Coulter counter, referring to the idea of blood cell counts through a capillary in the presence of a conductive electrolyte, coupled to a pico-ammeter to measure the pulses.

(11) The most significant advantage of a nanopore system aside from its small size and speed, however, is the lack of need for PCR amplification, as only 200 ng or less of viral material is required for analysis. As a strand of DNA is passed through a nanopore, the current is changed as the bases G, A, T and C pass through the void in different combinations. In addition, the system is the ability to continuously sample material.

(12) Graphene sheets are now being tested by several investigators, that improve both the resolution and speed in which nucleotide sequences can be sampled using nanopore technology. This will allow sampler reduced in size and higher sensitivity of the sequencer to incoming genomic samples.

(13) Improved Nanopore Genetic Sequencing in Vacuo

(14) Nanopore sequencing resolution can be improved by using graphene sheets in lieu of protein based nanopore structures. However, real-time DNA sequencing is currently a major challenge because longitudinal current detection cannot distinguish individual nucleotides due to the thickness of membrane (>10 bases) and the fast translocation of a single base.sup.2. In any nanopore system, an electrolyte is required to create an ion flow from one side of the pore to the other, dragging along the nucleotide strand with it. It is hereby proposed to use an ionic liquid as the electrolyte, or a silicone based diffusion pump oil seeded with an ionic liquid. The point of this approach is that the witness plate in the charge detection mass spectrometer can actually be the genetic sequencer in vacuo! In fact, it may be possible to create hundreds, if not thousands, of graphene nanopore sequencer ‘cells’, arranged in a matrix such that each cell can be selected for new genome sample processing depending on the desired type of virion being interrogated. The charged virions emitted from the charge detection mass spectrometric filter (we use the term charged particle as the virus is not an ion in the traditional sense), and electrostatically or magnetically deflected to the desired sequencing cell.

(15) This concept can be envisioned as a charged species equivalent to the old style cathode ray tubes used in early televisions and computer monitors. In those devices, an electron beam was continually deflected and scanned across a phosphor-coated screen to complete a raster. Similarly, one can deflect charged virions to any desired target for immediate analysis or sequesterization for subsequent laboratory study.

(16) Improved Nanopore Nucleic Acid Sequence Correlation

(17) One issue with nanopore genetic sequencing is that the nucleic acid strand passes through the pore very quickly, such that the resulting charge fluctuation due to individual base pairs may not yield as sharp discrete steps as would be the case if the strand passed through the pore more slowly. This reduces current resolution and thus nucleic acid identification accuracy. At present, this is dealt with by repetitive scans that are averaged or correlated over time to produce the desired current change indicative of the target sequence. One possible solution to this problem is the use of alternating current or AC instead of a DC bias in the electrolyte solution across the nanopore. By varying the frequency, polarity, and duty cycle of the applied potential, the target strand can be repetitively moved back and forth through the nanopore, permitting the requisite iterative sweeps required for acceptable signal averaging in far less time than using DC.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference Numerals

(18) FIG. 1: 10 Represents a conductive fluid capable of electrospray operation which may be volatile or non-volatile 20 Denotes a needle or tube or capillary for containing and supporting an electrospray 30 Is a Taylor Cone 40 Is a jet of fluid from an electropsray 50 is a dispersion of electrospray droplets 60 represents the coulomb explosion of electrospray droplets 70 represents desorbing electrospray droplets 80 represents bio-aerosol species in air being attracted to desorbing electrospray droplets 90 is a collection plate or witness plate for collection of charged bio species 100 is a collection plate or surface that is preferably a nanopore target 110 represents a power supply providing an electrical potential sufficient to yield a Taylor Cone from a needle or capillary 20 or paperspray source

(19) FIG. 2: 40 represents an electrospray source in totality encompassing elements 20,30,40, and 110 from FIG. 1 80 represents incoming ambient bioaerosols just before contact with electrospray droplets as in reference 80 120 is a capillary tube 130 are skimmers used to streamline the incoming charged species into a singular beam or linear stream of charged species 140 is an image charge detector tube 150 is a collection plate or target 160 is a picoammeter coupled to analysis device such as a computer

(20) FIG. 3: 170 Is a power supply for creating a difference of potential on either side of a nanopore 180 represents a strand of DNA, RNA, or any nucleotide sequence, or a protein or other biological molecule 190 is a nanopore, preferably in a sheet of graphene 200 is an electrolyte solution

(21) FIG. 4: 80 represents a strand of DNA, RNA, or any nucleotide sequence, or a protein or other biological molecule 200 is an electrolyte solution

(22) FIG. 5: 210 is a diagram of a cathode ray tube 220 is a diagram of a charged particle delivery system 230 is the sample inlet 240 is an electrospray source 250 is an electrostatic focusing system for the electrospray 260 represents horizontal electrostatic deflection plates 270 represents vertical electrostatic deflection plates 280 represents a nanopore detection target 290 represents a beam of charged bio-species, such as organic ions, viruses, bacteria, prion, protein, or nucleic acid

(23) FIG. 6: 300 Denotes the starting point of one scan preferred raster scanning of charged bio-species into well targets for preferred nanopore analysis 310 Denotes the end point of one scan preferred raster scanning of charged bio-species into well targets for preferred nanopore analysis

(24) FIG. 7: 320 Denotes ambient air laden with bio-aerosol species sample introduction 330 Denotes 1.sup.st stage heavy fraction filtration 340 Denotes electrospray charging of ambient bio aerosol species 350 Denotes second stage electrostatic filtration of bio species 360 Denotes charge detection mass spectroscopy and gating electrodes 370 Denotes lysing, extraction, and nanoporesequencing 380 Denotes data extraction step 390 Denotes Data transmission or migration to data cloud, internet, or network 400 Denotes application of predictive modeling algorithm

DETAILED DESCRIPTION OF DRAWINGS

(25) FIG. 1 is a diagram of the electrospray capture process for biological trace species from the air. The process begins with the introduction of a conductive fluid driven by either capillary action or hydrostatic force into an electrically conductive needle 20, where a difference of electric potential is applied between the needle 20 and a target 90 using a power supply 110 capable of creating a Taylor Cone 30 from which emerges a jet of fluid 40 which as the fluid evaporates 50, results in desorbing droplets 70, said droplets carrying a surface charge such that ambient biological species 80 are attracted and absorbed by aforementioned desorbing droplets 60, until only charged biological trace species remain and are deposited onto the opposing electrical pole target 90, which is preferably a graphene nanopore with an electrolyte on either face, said electrolyte preferably being a low volatility ionic liquid that will not evaporate under any level of vacuum, to which a difference of electrical potential has been applied.

(26) FIG. 2 illustrates the preferred embodiment of the invention, a charge detection mass spectrometer with a target that consists of a nanopore detector 150. The electrospray source 40 emits desorbing electrospray droplets to which ambient polar or polarizable biological trace species in the air are attracted 80 and absorbed by the droplets, resulting in discrete charged particles. The charged biological particles pass through a capillary tube 120 and into a succession of progressively pumped regions separated by skimmers 130 and through an image charge detector tube 140, said image charge of charged biological trace species being detected and amplified by a picoammeter and computer 160, before encountering end target 150 that is preferably a nanopore device capable of both lysing biological trace species and allowing a charge fluctuation of nucleic acid or other biological molecular group to pass through said nanopore, allowing identification of each molecular or atomic group or species.

(27) FIG. 3 discloses a nanopore nucleotide detection configuration. The nanopore, preferably a nano scale hole through a graphene sheet 190, where an electrolyte 200 is in contact with on either side, whereby a difference of potential 170 has been applied, such that a biological molecule, preferably a nucleotide sequence such as DNA 180, can be driven by ionic flow and pass through the aforementioned nanopore 190.

(28) FIG. 4 is a close up picture of a graphene nanopore 190 where a nucleotide sequence such as DNA 180 can pass through said nanopore 190, so driven by the flow of ions in an electrolyte solution in contact with both sides of the graphene sheet, insulated by a dielectric 200 from both sides of the nanopore.

(29) FIG. 5 is of two devices, a charged particle delivery system 220, and a cathode ray tube or CRT 210, upon which it is based. In a CRT, an electron gun emits electrons from a heated filament, which are electrostatically focused and then deflected using electrostatic or electromagnetic means such that a scan of the phosphor coated screen can be scanned in a successive raster pattern. The phosphor screen illuminates in the visible spectrum when high energy electrons strike its surface.

(30) In the preferred charged particle delivery system 220, electrospray charged bioaerosol trace species 230, devoid of solvent or aqueous solution and introduced into a preferably partial pressure region, passing through a charge detection mass spectrometer tube 240, are electrostatically focused 250, and then deflected by horizontal 260 and vertical 270 plates, and scanned in a raster pattern or simply directed to a specific target on a nanopore detection surface 280, said charged bio-aerosol consisting of particulates or virions or nucleic acid or bacteria, or protein or peptide or any bio-species 290.

(31) FIG. 6 is a raster pattern with a beginning point 300 and a terminal point 310, before the scan is retraced to the next level below or down next to and parallel to the preceding row of scan targets. Each target preferably represents an individual graphene ionic liquid nanopore well. Successive wells permit selective analysis depending upon the composition of the charge detection bio-particulate sample.

(32) FIG. 7 is a flowchart of the preferred bio-aerosol and infectious agent air sampling system. Ambient air containing bio-species 320 is drawn in using a fan or other fluid movement device, where heavy fractions 330 are separated using aerosol separation techniques such as moving through a sharp bend. The bioaerosols are electrically charged after undergoing interactions with an electrospray plume 340. Electrostatic separation 350 further refines the charged particulates, Charge detection mass spectrometry provides charged bio-aerosol or pathogen image charge and m/z value, after which lysing, nucleic acid extraction, and genetic sequencing is performed 370, after which the fusion of data analysis and extraction 380 is performed, whereby said data is uploaded to the internet or cloud or other network 390, for subsequent predictive modeling 400.