TAXONOMIC IDENTIFICATION OF MICROORGANISMS, PROTEINS AND PEPTIDES INVOLVED IN VERTEBRATE DISEASE STATES
Background Of The Invention
The present invention relates to a method and
apparatus for the taxonomic identification of microorganisms,
and more particularly to the taxonomic identification of
pathogenic microorganisms.
Pathogenic microorganisms, particularly pathogenic
bacteria which either occur naturally or which have acquired
virulence factors, are responsible for many of the diseases
which plague mankind. Many of these bacteria have been proposed
as biowarfare agents in the past. In addition, there is also
the risk and likelihood that nonpathogenic microbes could also
be used after genetic manipulation (e.g. , Escherichia coli
harboring the cholera toxin) .
Typical pathogenic bacteria include those responsible
for botulism, bubonic plague, cholera, diphtheria, dysentery,
leprosy, meningitis, scarlet fever, syphilis and tuberculosis, to mention a few. During the last several decades, the public perception has been one of near indifference in industrialized
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nations, principally because of successes that have been
achieved in combating these diseases using antibiotic therapy.
However, bacteria are becoming alarmingly resistant to
antibiotics. In addition, there have been recent revelations of
new roles that bacteria perform in human diseases such as
Heliobacteria phylori as a causitive agent of peptic ulcers,
Burkholderia cepaccia as a new pulmonary pathogen and Chlamydia
pneumoniae as a possible trigger of coronary heart disease.
Apart from those pathogens, various socioeconomic changes are
similarly contributing to the worldwide rise in food-borne
infections by bacteria such as Escherichia coli . Salmonella
spp . , Vibrio spp . , and Campylobacter jejuni .
Potential infections are also important considerations
in battlefield medicine. A number of bacterial pathogens,
including Bacilis anthracis and Yersininia pestis and their
exotoxins, have been used as weapons in the past. And, as
noted, there is always the increasing risk that nonpathogenic
microbes can be engineered to be pathogenic and employed as
biowarfare agents.
Pathogenic microorganisms are also of concern to the
livestock and poultry industries as well as wildlife management. For example, Brucella abortis causes the spontaneous abortion of
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calves in cattle. Water supplies contaminated with exotoxin-
producing microorganisms have been implicated in the deaths of
bird, fish and mammal populations. More recently, mad cow
disease has been traced to the oral transmission of a
proteinaceous particle not retained by filters. Thus, there is
clearly a need for the rapid and inexpensive techniques to
conduct field assays for toxic proteins and pathogenic
microorganisms that plague animals as well as humans.
As a general proposition, bacterial contamination can
be detected by ordinary light microscopy. This technique,
however, is only of limited taxonomic value. The investigation
and quantitation of areas greater than microns in size are
difficult and time consuming. Many commercially available
systems rely on the growth of cultures of bacteria to obtain
sufficiently large samples (outgrowth) for the subsequent
application of differential metabolic tests for species (genus)
identification. However, techniques requiring bacterial
outgrowth may fail to detect viable but nonculturable cells. To
the contrary, the growth media employed may favor the growth of
bacteria with specific phenotypes .
More sensitive and more rapid typing schemes are described in "Strategies to Accelerate the Applicability of Gene
6UBSπiUTESHEET(RULE26)
Amplification Protocols for Pathogen Detection in Meat and Meat
Products" by S. Pillai and S.C. Ricke and "Molecular Approaches
for Environmental Monitoring of Microorganisms" by R.M. Atlas, G.
Sayle, R.S. Burlage, and A.K. Be . Those techniques employ the
polymeric chain reaction (PCR) for amplification of bacterial
DNA or RNA, followed by nucleic acid sequencing to detect the
presence of a particular bacterial species. Such general
amplification and sequencing techniques require technical
expertise and are not easily adaptable outside of specialized
laboratory conditions. Moreover, such PCR methodology cannot
indicate whether the target bacterium was viable; this technique
provides a positive analysis whenever an intact target nucleic
acid sequence is detected.
Another approach utilizes immunochemical capture as
described in "The Use of Immonological Methods to Detect and
Identify Bacteria in the Environment" by M. Schlotter, B. Assmus
and A. Hartmann Biotech. Adv. 13, 75, followed by optical
detection of the captured cells. The most popular immunoassay
method, enzyme-length immunosorbent assay (E ISA) , has a
detection limit of several hundred cells. That is well below
the I.D. so of extremely infectious bacteria such as Shigella flexneri . Piezoelectric detection techniques, such as those described by "A Piezoelectric Biosensor for isteria eUBSnTUTE8HEET(RULE26)
Monocytogenes" by M.B. Jacobs, R.M. Carter, G.J. ubrano and G.G.
Guilbault, are even less sensitive having a detection limitation
of about 5 x 105 cells. A recent report entitled "Biosensor
Based on Force Microscope Technology" by D.R. Baselt, G.U. Lee
and R.J. Colton describes the use of an atomic force microscope
(AFM) to detect immunocaptured cells. Like other immunoassay
techniques, viable cells cannot be discriminated from dead cells
because cell capture and detection is predicated on the presence
of an intact bacterial antigen. Im unoassays are also presently
used in the trace analysis of peptides and proteins.
Moreover, the prior art has made extensive use of
immobilized antibodies in peptide/protein/microorganism capture.
Those techniques likewise involve significant problems because
the antibodies employed are very sensitive to variations in pH,
ionic strength and temperature. Antibodies are likewise
susceptible to degradation by a host of proteolytic enzymes in
"dirty" samples. In addition, the density of antibody molecules
supported on surfaces (e.g.. microwell plates or magnetic beads)
is not as high as is frequently necessary.
Medical and military considerations call for better toxin and pathogen detection technologies. Real-time assessment
of battlefield contamination by a remote sensing unit is
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necessary to permit and facilitate rapid diagnosis to permit
appropriate counter-measures. A microbe/toxic protein sensor
useful in such situation requires the ability to globally
discriminate between pathogens and nonpathogens as well as
discriminate between viable cells (including spores) and dead
cells. In addition, such techniques require high sensitivity
when ten or fewer cells are present and analysis that can be
completed in the field in less than 15 minutes. In cases such
as this, even in pyrolysis, followed by mass spectral analysis
of volatile cell components (e.g. , fatty acids) would be
extremely difficult and hence impractical for routine assays.
Such techniques should be able to recognize pathogens and
provide some assessment of strain virulence or toxigenicity .
In copending application Serial No. 559,043, filed
June 3, 1996, the disclosure of which is incorporated herein by
reference, there is described a method and apparatus for sensing
the presence of microbes on a non-living surface which is
particularly well -suited to detect the presence of microbes in
meat, poultry and like food products. In accordance with the
system described in the foregoing copending application,
microbes which may be present on non-living surfaces such as
meat and poultry are subjected to electromagnetic energy having
wavelengths greater than about 350 nm. The electromagnetic
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radiation excites the microbial cells present on the surface to
emit electromagnetic energy (i.e. , fluoresce) having a
wavelength greater than that of the excitation wavelength. Any
microbial cells present on the surface containing reduced
pyridine nucleotides such as nicotinamide adenine dinucleotide
(NADH) will emit a characteristic fluorescence signal. The
presence of living microbial cells is determined by sensing both
the fluorescence from the cell respiration and electromagnetic radiation reflected or scattered by the surface.
While the system disclosed in the foregoing
application represents a significant advance in the art in
detecting the presence of living microbial cells, it cannot be
used as such to taxonomically evaluate the microbes present on
the surface, or identify proteinaceous toxins or peptide
hormones of pathophysiological importance to vertebrate animals,
including humans, livestock, poultry and wildlife.
It is accordingly an object of the present invention
to provide a method and apparatus for taxonomically evaluating
microbes, proteins or peptides which overcome the foregoing
disadvantages .
It is a more specific object of the invention to
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provide a method and apparatus for taxonomically evaluating
microbes, proteins and peptides which have the capability of
discriminating between pathogens and nonpathogens and viable
cells from dead cells and can be likewise used to identify
proteins or peptides.
It is yet another object of the invention to provide a
method and apparatus for taxonomically evaluating microbial
cells characterized by high sensitivity (ten to one hundred
cells) and which can taxonomically identify microbes under field
conditions and can likewise be used to identify trace amounts of
proteins or peptides.
Summary Of The Invention
The concepts of the present invention reside in a
method and apparatus for the taxonomic identification of
microorganisms in which microbes are captured through the
binding of microbial receptors to specific ligands tethered to a
surface, and the electromagnetic radiation is used to determine
the presence of metabolites or other characteristic biomolecules
for the detection of the presence of the captured microorganisms
in accordance with the practice of the invention, a microorganism-containing sample is contacted with a sensor chip,
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the sensor chip having a patterned area on its surface
containing a plurality of sections, with each section having
bonded thereto a ligand capable of bonding to a specific
microbial receptor. The receptor may be, for example, a protein
residing in the outer membrane of the microbial cell, pilus or
flagellum which is exposed to the aqueous environment
surrounding a cell. In accordance with the concepts of the
present invention, the same receptor likewise forms the basis
for the detection of peptides and proteins of pathological
interest .
Electromagnetic radiation is directed onto the surface
of the sensor chip to excite sections of the sensor chip to
determine which of the sections of the sensor chip contain a
microorganism binding to the ligand on that particular section.
By determining which sections have a microorganism bound to a
ligand, it is possible to taxonomically identify the
microorganism contained in the sample as a function of the
combination of different ligands which have a microbe bound
thereto.
Thus, the method of the present invention does not depend on classical antigen-antibody recognition. On the
contrary, the concepts of the present invention make use of
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relatively inexpensive reagents in the capture of
microorganisms, peptides or proteins contained in the sample.
The sensor chip employed in the practice of the
present invention are preferably formed from a suitable support
material such as glass or non-reactive plastic substrates such
as polystyrene and polymethylmethacrylate . The sensor chip is
formed of a patterned array defining a plurality of sections on
the surface of the sensor chip, and each section has bonded
thereto a different ligand capable of molecularly recognizing a
specific peptide, protein or microbial receptor and hence the
microbe itself. The ligand for pathogen/peptide/protein capture
bonded to the surface of the sensor chip can and should be
varied. In general, such ligands may be characterized as heme,
siderophores , oligosaccharides and anti-adhesion peptides
capable of capturing a wide variety of microorganisms, toxic
proteins and peptides. Those ligands can thus be immobilized or
bonded to the surface of the sensor chip by means of organic
coupling agents having the capability of reacting with the
surface of the sensor chip itself and also having the capability
of reacting with the ligands whereby the coupling agent
establishes a chemical bond or "tether" between the surface of
the sensor chip and the ligand capable of reaction with a variety of different microorganisms, proteins and/or peptides. ESπTUTESHEεr(RU E26)
Particularly useful in the bonding of the ligands to glass
sensor chip substrates are the organosilanes having 1-3 readily
hydrolyzable groups attached directly to the silicon atom and a
functional organic group also attached to the silicon atom, the
functional group being capable of reaction with the ligand.
In the preferred practice of the invention, the
patterned array of the sensor chip is preferably positioned on
the surface thereof in a pattern such as rows of sections. Each
one of the sections, in the preferred practice of the invention,
is exposed to electromagnetic radiation to excite captured
protein, peptide or biomolecule present in the captured microbes
having characteristic emission fluorescence. That fluorescence
can then be detected by a suitable apparatus for detecting
electromagnetic radiation and converting that radiation into an
electrical signal as an indication of whether or not a
particular section has a microorganism, protein or peptide
bonded to the ligand for that section. In the preferred
practice of the invention, the probe, which may be a probe like
that described in the foregoing copending application, can be
sequentially positioned to direct electromagnetic radiation to
each of the sections in turn. Alternatively, each section of
the sensor chip can be scanned simultaneously. The output of the fluorescence detector can then be converted to an electrical
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signal indicative of those sections having a microorganism,
protein or peptide bonded thereto. The microorganism, protein
or peptide present can thus be identified by examining which of
the sections have captured the species of interest and which
have no .
Thus, the present invention can be rapidly used to
identify microorganisms without the need for growing a culture
of the microorganism and then microscopically examining the
culture thus produced. Likewise, low levels of toxic proteins
or peptide hormones can similarly be identified. It is also
unnecessary to employ enzymes or antibodies in the capture of
microbial metabolites as is often used in the prior art.
Brief Description Of The Drawings
Fig. 1 is a schematic illustration showing the sensor
chip employed in the practice of the invention, illustrating the
different sections contained on the face thereof having
different ligands attached to each section.
Fig. 2 is a schematic illustration of a multiple
element detection system which can e used in the practice of the present invention.
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Fig. 3 is a graph illustrating the fluorescence
excitation (EX) and emission (EM) spectrum of a sample of meat
with and without E coli contamination and meat with fat having
no E coli contamination. Contamination was approximately 103
cells/cm2.
Fig. 4 is a graph illustrating fluorescence excitation
(EX) and emission (EM) spectrum of tryptophan (Trp) , tyrosine
(Tyr) and calcium dipicolinate (DP) in Bacillus cereus spores.
Fig. 5 illustrates schematically a system embodying
the present invention for scanning a sensor chip in the practice
of the invention.
Detailed Description Of The Invention
In the practice of the present invention, a sample
containing an unknown analyte microorganism, protein or peptide
is first contacted with the sensor chip. The sensor chip is
illustrated in Figure 1 of the drawings and is formed of a
substrate 8 such as a glass slide having a series of sections
formed thereon, each of which having a series of sections 1
through 70 on the surface thereof. Each section has a different ligand bonded thereto so as to be capable of binding to specific
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analytes. The ligands are capable of binding to the analyte for
capture and the presence of the captured analyte is detected
using the fluorescence detection system disclosed and claimed in
copending application Serial No. 659,043, filed June 3, 1996.
Thus, the ligand of each of the sections of the sensor chip 8
has the capability of capturing a specific microorganism,
peptide or protein.
In the preferred practice of the invention, the
ligands used in the present invention are taken from the group
of heme compounds, siderophores, oligosaccharides and peptides.
As is well known to those skilled in the art, animal
pathogens generally possess heme uptake capability, and thus
heme compounds can be used to capture a number of pathogenic
species. In addition to heme compounds, other ligands in the
form of high-affinity iron chelators, generally referred to as
siderophores, can also be used to capture many strains of
pathogenic bacteria. Included among such siderophores are
alcaligin, mycobactins, pyochelin, staphyloferrin, vibriobactin and yersiniabactin.
In addition to heme compounds and siderophores,
eukaryotic surface epitopes (peptides or carbohydrates) which su8SimjτesH£er(R E26)
are recognized by microbial cell receptors, can likewise be used
as ligands in the practice of the present invention. These
ligands include commercially available oligosaccharides as well
as those available by chemical synthesis. Other
oligosaccharides and their affinity to pathogens from various
microorganisms are described by Karlsson "Microbial Recognition
of Target Cell Glycoconjugates" , Structural Biology, 1995, 5:
622,635, the disclosure of which is incorporated herein by
reference .
The characteristics of a number of bacterial species
along with the diseases caused by such bacteria and their
binding characteristics with siderophores, oligosaccharides and
hemin are set forth in Table I. These characteristics can be
used in the capture and identification of such species.
Peptide ligands can be produced by affinity panning of
libraries of oligopeptides displayed on bacteriophages or on
Escherichia coli flagella. Such ligands are useful in the
capture of soluble proteins and peptide hormones as well as
microorganisms .
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Table 1
Table 1 (cont)
Toxins that contain at least one tryptophan or several
tyrosines per molecule can be detected by tryptophan/tyrosine
fluorescence after capture using a tethered peptide (produced by
biopanning a library of peptides) . A variety of microbes,
including algae, fungi, protozoans, and bacteria export
exotoxins that are amenable to detection using this technology.
Selected tissues of a variety of higher plants yield toxic
proteins. A variety of animals, including reptiles, amphibians,
marine invertebrates, scorpions, spiders, and insects, produce
toxic proteins and peptides as well. The following list
contains examples of toxic proteins and peptides that can be
captured and detected using the technology described herein.
Ridinus communis (castor bean) ricins
Apis mel lifera (honey bee) mellitin
Latrodectus mactans (black widow spider) α-latrotoxin
Agelenopsis aptera (funnel web spider) ra-agatoxin TK Bunodosoma granulif era (sea anemone) K+-channel -blocking toxin
Bungarus mul ticinctus (krait) β-bungarotoxin Naja naja atra (Formosan cobra) cobratoxins
Bacteria:
Bacillus anthracis anthrax toxins (all components
Clostridium botulinum botulinum toxins Vibrio Cholerae cholera toxin Clostridium perfringens α-toxin
(phospholipase C)
Corynebacterium diphtheriae diphtheria toxin Escherichia coli heat-labile enterotoxin Bordetella pertussis pertussis toxin Shigeila dysenteriae Shiga toxin Staphylococcus aureus toxic shock syndrome
Clostridium tetani tetanus toxin Yersinia pestis YopE Helicobacter pylori vacuolating cytotoxin A
Examples of human bioactive peptides (including
peptide hormones) that can be detected using tryptophan/tyrosine fluorescence :
adrenocorticotropic hormone (ACTH) bombesin gastrins gastrin-releasing peptide (GRP) neuropeptide Y (NPY) luteinizing hormone releasing hormone (LH-RH) β-melanocyte stimulating hormone parathyroid hormone (PTH) somatostatin endothelins
The various ligands are preferably tethered to a
substrate by means of organic coupling agents which are
themselves well known to those skilled in the art. When using a
glass substrate for the sensor chip, it is frequently preferred
to employ, in the practice of the present invention,
organosilane compounds having the following general structure:
I R2 - Si - R4 I R3
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wherein R. through R3 are each selected from the group consisting
of hydrogen, alkyl groups containing 1 to 6 carbon atoms, aryl
groups containing 6 to 12 carbon atoms and alkoxy groups
containing 1 to 4 carbon atoms, with at least one of Rx, R2 and
R3 being an alkoxy group. R4 is an organic group containing at
least 3 carbon atoms and also containing a functional group
capable of reaction with the ligand. Without limiting the
invention, suitable organic groups are polyamines and polyethers
containing 3 to 30 carbon atoms. Also suitable for use in the
practice of the invention are coupling agents containing other
functional groups such as epoxy groups, amino groups and
unsaturated functional groups, OH groups, thiol groups and the
like, which are capable of reaction with the various ligands.
Without limiting the invention as to theory, it is believed that
the ligand reacts with the functional group, preferably a
terminal functional on the organosilane compound while the
readily hydrolyzable alkoxy group attached directly to the
silicon atom has the capability of reacting directly with the
surface of the glass substrate of the sensor chips. Thus, the
ligand is tethered to the surface of the glass through the
coupling agent.
Thus the ligand tethered to the glass surface may be illustrated by the following:
Glass-0-Si-(CH
2)
3-NH-(CH
2)
3-NH-(CH
2)
5-NH-(CH
2)
12-NH-(CH
2)
5-NJ-LIGAND
The chemical reactions used in tethering ligands to the surface
of the sensor chip are known to those skilled in the art and are
described in the literature. Such reactions may be found in
G.T. Hermanson, Bioconiugate Techniques. San Diego: Academic
Press, 1966; Hansson et al . , "Carbohydrate-Specific Adhesion of
Bacteria to Thin Layer Chromatograms: A Rationalized Approach to
the Study of Host Cell Glycolipid Receptors", Analytical
Biochemistry, 146, 158-163 (1985); and, Nilsson et al . , "A
Carbohydrate Biosensor Surface for the Detection of
Uropathogenic Bacteria", Bio/Technology, 12, 1376-1378, December
1994.
Illustrative of such reactions are those used to
tether ferroxamine as a ligand to the surface of a glass sensor
chip. In the first stage, a glass surface containing free
hydroxyl groups is first reacted with a 2% solution of gamma-N-
(aminopropyl) -gamma-aminopropyltrimethoxysilane to attach the
silane to the glass surface:
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Glass-OH+(CH3O)3-Si-(CH2)3-NH-(CH3)3-NH2→
( i :
Glass-O-Si-(CH2)3-NH-(CH2)3-NH
The product of that reaction can then be reacted with
glutaraldehyde at a pH of about 8 to form the corresponding
aldehyde :
Glass-O-Si-(CH2)3-NH-(CH2)3-N=C-(CH2)3-C-H ( 11 ;
The aldehyde, in turn, can be reacted with a diamine:
H2N-(CH2)12-NH2 (III)
Glass-O-Si-(CH2)3-NH-(CH2)3-N = C-(CH2)3-C = N-(CH2)12-NH2 ( IV)
H H
Next, the product of the preceding reaction is reacted
with glutaraldehyde to introduce a (terminal) aldehyde group:
SUBSHTUTESHEET(RULE2S)
Glass-O-Si-(CH2)3-NH-(CH2)3N = C-(CH2)3-C = N-(CH2)12-N = CH-(CH2)3-CHO (V)
H H
which can then be reduced using NaCNBH3 to yield:
Glass-O-Si-(CH2)3-NH-(CH2)3-NH-(CH2)5-NH-(CH2)12-NH-(CH2)4-CHO (VI)
The foregoing silane coupling agent bonded to the
surface can then be derivatized by reaction with deferrioxamine
B (or DFA) at an alkaline pH to yield:
Glass-O-Si-(CH2)3-NH-(CH2)3-NH-(CH2)5-NH-(CH2)12-NH-(CH2)4-CH = N-DFA (VI I )
I I
H
The DFA can then be complexed with Fe by reaction with a ferrous
salt in aqueous medium to form the ligand.
As will be appreciated by those skilled in the art,
many other techniques can likewise be used to tether an
appropriate ligand to the surface of the sensor chip. For
example, thiol-terminated peptides can be tethered to the
surface of a glass sensor chip using similar reactions. For
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example, IV above can be reacted with N- (gamma-
alemidobutyryloxy) succinimide ester (GMBS) to form the
following derivative:
That derivative can, in turn, be sequentially reacted with the thiol-terminated peptide to form the corresponding
peptide terminated compound. That, in turn, can, if desired, be
reduced using sodium cyanoborohydride to yield the following peptide ligand tethered to the glass surface:
Similarly, the same overall reaction scheme can
likewise be used to tether hemin to the glass surface of the sensor chip. Hemin, also known as ferriprotoprophyrin IX, can
be reacted with N-hydroxysuccinimide to form the hemin di (N-
hydroxysuccinimide) ester. That diester can then be reacted
with the product IV referred to above which has been previously reduced with sodium cyanoborohydride to form the following
tethered ligand:
Thus, as described above, a different ligand is tethered to each of the sections of the sensor chip. The sensor
chip is then contacted with a sample containing an unknown
organism, protein or peptide whereby specific ligands on the
surface of the chip bind to specific analytes, selectively
capturing them. The sensor chip is then subjected to electromagnetic radiation using the equipment described in
Serial No. 659,043 so that each section of the sensor chip is
exposed to an appropriate wavelength of electromagnetic
radiation to excite fluorescence characteristic of the presence
of bound analytes. Appropriate -fluorescence signals generated by metabolites or other biomolecules specific to pathogenic
microorganisms on exposure to electromagnetic radiation are set
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forth in Table 2. Note NAD[P]H is only present in viable or
respiring cells; calcium dipicolinate as a significant presence
in spores of the order of about 15% but is otherwise rare in
nature. The relative signals as measured in bacteria and spores
are shown in Figs. 3 and 4, respectively. Any proteinaceous
toxin or peptide hormone that contains tryptophan or tyrosine
will also generate fluorescence signals as noted in Table 2 for
these fluorophores .
The sensor is illustrated in Fig. 5 of the drawing in
schematic form, and includes a source of light 10 having the
desired wavelength. As will be appreciated by those skilled in
the art, the source of electromagnetic radiation 10 can be any
of a variety of devices producing UV light having a wavelength
less than about 400 nm. For example, the source of
electromagnetic radiation 10 can be a laser or various types of
lamps emitting electromagnetic radiation generally within the UV
range .
The wavelength of light is chosen by an excitation
filter 12, preferably in the form of a narrow band-width filter
passing electromagnetic radiation having wavelengths within the
desired range. For example, when seeking to excite NADH, the
filter 12 should be one which emits electromagnetic radiation
©U8SimJΪES- Er(RULE2S)
within the range of about 350 to 390nm and preferably with a
peak of about 366nm.
Positioned to receive the light emitted from the light
source 10 through filter 12 is a conduit for electromagnetic
radiation 14, preferably in the form of a fiber optic element or
bundle of elements, capable of conducting the electromagnetic
energy passing from the source 10 through the filter 12. The
fiber optic bundle terminates in a probe 16, which can be a
probe used to successively scan each of the sections of the
sensor chip, as in a rastor scan or in one element of a multi¬
element probe system with one element for each section.
Also contained within the probe are a pair of
additional fiber optic elements or bundles 20 and 22 illustrated
in Fig. 2, positioned to transmit electromagnetic radiation from
the sensor chip 8 for detection. In the illustrated embodiment,
fiber optic element 20 conveys electromagnetic energy from the
sensor chip 8 to an emission filter 24; that filter is chosen to
pass electromagnetic radiation having the wavelength of the
fluorescence generated in the microbial cells, peptides or
proteins present on each of the sections of the sensor chip 8.
It is also desirable, in the practice of the
SUBSmumSHEEr(RULE26)
invention, to use another filter 26 associated with fiber optic
element 22 which passes only electromagnetic radiation having substantially the same wavelength as the electromagnetic
radiation directed to the sensor chip through fiber optic
element 14. Thus, filter 26 should pass electromagnetic
radiation within the same range as that used for excitation.
The choice of excitation and emission filters is set forth in
Table 2. The filter combinations can be easily changed by
mounting the filters on a rotating wheel or sliding mechanism to
provide detection of all of the compounds in Table 2.
The apparatus shown in Fig. 5 also includes a pair of
detector elements 28 and 30 which detect electromagnetic
radiation passing through filters 24 and 26, respectively. As
will be appreciated by those skilled in the art, the detectors
28 and 30 are elements sensitive to electromagnetic radiation,
converting that radiation into an electrical signal which is
proportional to the intensity of the radiation presented to the
detectors through filters 24 and 26, respectively. Conventional
devices including photodiodes, photomultiplier, tubes, video
cameras, charge-coupled devices as well as other detectors may be used.
Because the electromagnetic radiation passed through
the associated filter 24 is essentially limited to the
wavelength of the electromagnetic energy associated with the
fluorescence of a specific metabolite or other biomolecules, the
amount of fluorescence due to that metabolite passing through the filter 24 from the sensor chip 8 and detected by the
fluorescence detector 28 is related to the amount of that
metabolite present and thus to the number of microorganism,
protein molecules or peptide molecules present. The
electromagnetic radiation passed through fiber optic element 22
is filtered to pass only electromagnetic radiation having a
wavelength substantially the same as that of the electromagnetic
energy directed into the sensor chip for that particular
section; detector 30 measures only the reflected radiation from
the sensor chip. Both detectors 28 and 30 thus convert the
electromagnetic energy to a corresponding electrical signal, and
the signal indicative of the presence of analytes of interest is
determined by dividing the amount of electromagnetic radiation
passing through filter 24 to detector 28 (representing the
fluorescence of any captured species present on that section of
the sensor chip) by the electromagnetic radiation passing
through filter 26 to the detector 30 (representing the reflected
electromagnetic radiation) . This signal is then normalized by
subtraction of this ratio for the blank section of the sensor chip. Thus, the signal may be represented by the difference
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between the fluorescence divided by the reflectance for a
section containing a captured analyte of interest minus the
fluorescence divided by the reflectance for a blank section.
An alternate system for scanning the sensor chip 8 is
illustrated in Fig. 2 of the drawing showing a sensor chip 8
which has been contacted with an analyte. Various sections 9
contain no captured analyte thereon while other sections 7 do
have a captured analyte. It is possible, and sometimes
desirable, to determine the presence of a bound analyte in each
of the sections of the sensor chip 8 simultaneously.
That can be done using a system described in Fig. 2
having a matrix support member 40 which includes a plurality of
probes 16 mounted therein, with the probes 16 mounted in the
matrix 40 being patterned to correspond to the pattern of
sections in the chip 8.
In the preferred embodiment, the matrix 40 includes
one probe 16 for each of the sections on the sensor chip 8,
although, as will be understood by those skilled in the art, either fewer or greater probes may be used under some
circumstances. The matrix is then positioned proximate to a
chip 8 which has been exposed to an unknown sample and each of
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the sections is exposed to electromagnetic radiation
simultaneously. Accordingly, each of the sections is
simultaneously examined by electromagnetic radiation to detect
the presence of captured analytes on each section of the chip 8.
The system of Fig. 2 otherwise operates in the same manner as
that described in Fig. 5.
By comparing fluorescence signals to reflected
signals, the system as described in detail in the foregoing,
copending application, normalizes the signals. That allows the
system to compensate for variations in the distance of the probe
from the surface of the chip 8 and variations between different
surfaces. As will be appreciated by those skilled in the art,
it is possible, and sometimes desirable, to either use multiple sources of electromagnetic radiation or to employ multiple
filters 12. In that way, the electromagnetic radiation directed
to a single section of the sensor chip 8 may be changed going
from one section of that chip to another section of the chip.
That technique thus permits different wavelengths of
electromagnetic radiation to be directed to different sections
of the sensor chip, depending on the ligand tethered to that
particular section of the sensor chip.
As will be appreciated by those skilled in the art, a
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variety of bacterial cell components or metabolites as well as
proteins and peptides exhibit intrinsic fluorescence when
illuminated by UV light. For example, NADH has been extensively
used in the study of various organisms and can be employed to
determine whether a particular microbial sample contains viable
or respiring cells (Fig. 3) . Similarly, tryptophan likewise
exhibits fluorescence indicative of spores, nonviable cells,
most protein toxins and many peptide hormones, while calcium
dipicolinate exhibits fluorescence indicative of the presence of
spores (Fig. 4) . Those fluorophores, their excitation and
emission frequencies in vivo as well as the indications they
provide are set forth in Table 2 :
In the practice of the present invention, a sample containing unknown microbes can be contacted with the sensor
chip whereby one or more receptors of the bacteria react with
various different ligands tethered to the various sections of
the chip. Then, the fluorescence of the chip can be measured
with the probe 16 for the purpose of detecting which of the
sections of the sensor chip have analytes bonded thereto. As
examples, Myobacterium siderophores can be used to capture
mycobacteria such as mycobacterium tuberculosis. Helicobacter
pylori can be captured using tethered N-acetylneuroaminyl-alpha- 2 , 3-galactose. The peptide:
GADRSYLSFIHLYPELAGAC
can be tethered, by means of the terminal systeine group to
suppress capture Staphylococcus aureus toxic-shock toxin- 1.
As indicated above, some of the analytes of interest
can be identified by determining the presence of a single
captured microorganism, protein or peptide. In other cases,
however, a series of two or more captured analytes of interest
is indicative of the identity of a particular analyte. As an example, consider a sensor chip having an area of three sections
along the horizontal axis and three sections along the vertical
axis as illustrated below:
As an example, the sections identified can be provided
with the following ligands tethered to each specific section as
set forth in the following table:
Section 10x10 Array Location Ligand
Al asialo Gml A2 hemin A3 pyochelin Bl GalNAcβGal B2 alcaligin B3 fibronectin peptide Cl diferric transferrin C2 staphyloferrin C3 ferrioxamine B
It has been found that Pseudomonas aeruginosa can be
identified as the microorganism when analytes are detected in
sections Al , A2 , A3, Bl, Cl and C3. Similarly, Klebsiella
Pneumoniae is detected when sections A2 , Bl, Cl and C3 have
analytes captured thereon, and Serratia marescens is identified
when sections A2 and C3 have analyte captured thereon. Similarly, Staphylococcus aureus can be identified when sections
A2, Bl, B3 , C2 and C3 contain analyte captured thereon.
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It will be understood that various changes and
modifications can be made in the determination, procedure,
formulation and use without departing from the spirit of the
invention, especially as defined in the following claims.
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