WO2022169991A1 - A face covering having a test strip for colorimetric monitoring of proteases and methods of detecting proteases - Google Patents
A face covering having a test strip for colorimetric monitoring of proteases and methods of detecting proteases Download PDFInfo
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- WO2022169991A1 WO2022169991A1 PCT/US2022/015141 US2022015141W WO2022169991A1 WO 2022169991 A1 WO2022169991 A1 WO 2022169991A1 US 2022015141 W US2022015141 W US 2022015141W WO 2022169991 A1 WO2022169991 A1 WO 2022169991A1
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- peptide
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- protease
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- G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
Definitions
- the COVID-19 pandemic is caused by the SARS-CoV-2 virus transmitted via aerosols or droplets expelled by speaking, breathing, or coughing.
- Non- pharmacological interventions e.g., mask-wearing and diagnostic testing
- Vulnerable populations do not just need testing — they need surveillance.
- Vulnerable populations such as residents (and caregivers) in group care homes, prisons, dialysis clinics, etc. are very susceptible to SARS-CoV-2 infection. Therefore, they should be tested frequently to quickly identify and isolate infected persons while notifying their contacts.
- An ideal test could be performed at the point- of-care by the person to be tested or their caregiver — it would be simple, affordable, reliable, and accurate.
- PCR and serology are the current gold standards to diagnose SARS-CoV-2 infection but have critical limitations. Serology only indicates prior exposure with few details on current viral activity. PCR requires a complex laboratory infrastructure, trained personnel, and multi-step sample preparation. Thus, widespread surveillance efforts will continue to suffer until these testing shortfalls are resolved.
- FIG. 1 shows a schematic diagram of one example of a face covering that incorporates a sensing test strip.
- FIG. 2 shows an exploded view of one example of the sensing test strip shown in Fig. 1.
- FIG. 3 A shows another exploded view of the sensing test strip shown in FIG. 2;
- FIG. 3B is a white-light image of a test strip affixed to the outside of a neck gaiter and another test strip offset parallel inside the mask; and
- Figs. 3C, 3D, 3E and 3F show the measured amylase level on the outside and inside test strips attached to different face coverings for a neck gaiter, for a cloth mask, a surgical mask, and a N95 respirator, respectively.
- Fig. 4A shows an example of an amylase-sensing test strip similar to that shown in Figs. 2 and 3 A but which includes an extra layer of forensic press paper underneath the polyester pad to act as a blue color agent; and Fig. 4B shows whitelight images of the amylase-sensing test strip before and after release of water.
- FIG. 5 A shows a schematic illustration of the aggregation of bis(/?- sulfonatophenyl)phenylphosphine-coated gold nanoparticles (BSPP-AuNPs) caused by the proteolytic hydrolysis of the intact peptide, where the net charge of intact peptide and its fragment is reversed;
- Fig. 5B shows that a colorimetric test coupled with face coverings having a lateral flow strip to indicate COVID infection in-situ (top); and Fig.
- 5C shows TEM images of the dispersed BSPP-AuNPs (top) and proteolysis-induced gold aggregates [bottom, (i)] and flocculated AuNPs redispersed using ionic surfactant additives [(ii)], due to the restored electrostatic repulsion.
- Fig. 6A shows a representative peptide ZY7
- Fig. 6B shows HPLC and ESIMS data showing that M pro selectively cleaves the ZY7 peptide at the C-terminus of glutamine (Q)
- Fig. 6C shows the color change (in gray scale) as a function of peptide concentration and time
- Fig. 6D shows DLS profiles of BSPP-AuNPs (3.8 nM, 100 pL) incubated with increasing concentrations of ZY7 parent peptide (darker circles) and ZY7 fragments (lighter circles);
- Fig. 6A shows a representative peptide ZY7
- Fig. 6B shows HPLC and ESIMS data showing that M pro selectively cleaves the ZY7 peptide at the C-terminus of glutamine (Q)
- Fig. 6C shows the color change (in gray scale) as a function of peptide concentration and time
- Fig. 6D shows DLS profiles of BSPP-Au
- Fig. 7A shows the ratiometric signal (Abseoo/Abs52o) collected from BSPP- AuNPs (3.8 nM, 100 pL) incubated with various amount of ZY7 parent and fragments;
- Fig. 7B shows the time progression of absorbance ratio in the enzyme assay, where a fixed amount of ZY7 substrate (50 pM) is incubated with increasing concentrations M pro (0-200 nM);
- Fig. 7C shows the ratio of Abseoo/Abs52o as a function of M pro concentration;
- Fig. 7D which shows the absorbance ratio as a function of M pro concentration in three biofluids, where MS7 (control) substrate (50 pM) is used.
- FIGs. 8 A, 8B and 8C show the operation window of M pro sensors based on NR.10, OR8, and YR9 peptide, which contains 0, 1, and 4 arginine residues in its aggregating sequence, respectively;
- Fig. 8D shows the M pro LoD of sensors based on four peptides of varying number of arginine;
- Fig. 8E shows the results of assaying an increasing molarity of GC376 (/. ⁇ ?., 0-1 pM) in the presence of constant amount of M pro (100 nM) and ZY7 substrate (50 pM);
- FIG. 8F shows a typical inhibition titration curve fitted with the Morrison equation for the competitive inhibitor, GC376, where the inset shows the chemical structure of GC376 inhibitor;
- Fig. 8G shows sensor activation by other mammalian proteins (100 nM), including bovine serum albumin (BSA), hemoglobin, a-amylase (100 U/mL), thrombin, and trypsin; and
- Fig. 8H shows the response of sensors based on ZY7 and DS 12 substrate to M pro or trypsin in Tris buffer.
- FIG. 9A show an illustration of the sensing test strip on a face mask and an exploded view of the sensing test strip
- FIG. 9B shows a white light image of the assembled sensing test strip, equipped with a BSPP-AuNP dispersion in the blister pack, ZY7 peptide on the test lane (left, dash box), and YF15 peptide on the positivecontrol lane
- Fig. 9C shows various concentrations of M pro in EBC (0-100 nM, with respect to 30 pL volume) tested for 10 min
- FIG. 9A show an illustration of the sensing test strip on a face mask and an exploded view of the sensing test strip
- FIG. 9B shows a white light image of the assembled sensing test strip, equipped with a BSPP-AuNP dispersion in the blister pack, ZY7 peptide on the test lane (left, dash box
- a colorimetric assay uses a stimuli-responsive peptide for detecting an enzyme.
- the assay includes a modular peptide having physicochemical properties that change in response to a presence of an enzyme of interest.
- the assay also includes a color reagent indicating a presence and quantitative level of the enzyme of interest when treated with the modular peptide via a visual readout.
- the quantitative level of modular peptide activation is related to a quantity of the enzyme of interest such that the colorimetric assay is applicable for purposes of inhibitor screening and diagnostic testing.
- one of the physicochemical properties is charge potential
- the modular peptide being a charge-switchable zwitterionic peptide having a general formula (Asp/Glu) «-(AA) x -(Arg/Lys) m , and being synthesized via a Fmoc-SPPS method, wherein an (Arg/Lys) m block of the modular peptide includes at least Arg and Lys units and an (Asp/Glu)neig block of the modular peptide includes at least Asp and/or Glu units.
- the color reagent includes negatively -charged- ligand functionalized plasmonic nanoparticles.
- the peptide when intact, is in a net neutral or negative charge state that has minimal interactions with the functionalized plasmonic nanoparticles, with n > m > 2.
- -(AA)r- is an amino acid sequence of varying length comprising natural and synthetic amino acids that is responsive to the enzyme of interest, the enzyme of interest being selected from the group consisting of a viral protease, a mammalian protease, or a combinations thereof
- installed ligands are phenyl phosphine derivatives selected from the group consisting of: diphenylphosphinobenzene sulfonate (DPPS), bis(p-sulfonatophenyl)phenylphosphine (BSPP), triphenylphosphine-3,3,3-trisulfonate (TPPTS), and combinations of the above.
- DPPS diphenylphosphinobenzene sulfonate
- BSPP bis(p-sulfonatophenyl)phenylphosphine
- TPTS triphenylphosphine-3,3,3-trisulfonate
- a charged ligand is installed on plasmonic nanoparticles by a ligand exchange or phase transfer reaction.
- plasmonic particles of the negatively- charged-ligand functionalized plasmonic nanoparticles are prepared from citrate- stabilized spherical gold nanoparticles by a Turkevich method.
- a negative charge on the peptide arises from aspartic acid, glutamic acid, or a combination of thereof and a positive charge arises from arginine, lysine, or a combination thereof.
- the charge-switchable zwitterionic peptide is used with gold nanoparticles under label free conditions.
- a mass concentration for ligand exchange is 0.5 to 2 mg of ligand per mL of nanoparticle volume.
- the enzyme of interest is a protease, a peptide fragment arising from protease breakdown being a positively-charged species that electrostatically interacts with gold nanoparticles.
- an electrostatic attraction of the gold nanoparticle induces a plasmonic coupling and color changes.
- a color change arising from nanoparticle aggregation occurs either after introducing the protease pre-mixed with the modular peptide to a dispersion of the gold nanoparticle or after introducing the protease to the modular peptide premixed with the gold nanoparticles.
- incubation of the protease with the modular peptide occurs at 37 °C for 0.5 to 3 hours, with a molar ratio of [peptide]: [protease] varying from 250 to 100,000.
- color changes at 10 min are recorded by eye or using a smart phone, an optical absorption spectrometer, or both.
- the gold nanoparticles are stable in buffers for no less than 24 hours.
- a sample to be analyzed contains biological samples.
- the biological samples are selected from the group including exhaled breath condensate, saliva, human plasma, urine and gingival crevicular fluid.
- the biological samples further include buffers.
- the buffers include dithiothreitol.
- a detection limit of the protease is at a first molar ratio of [peptide]: [protease] that shows an observable color change.
- an amount of gold nanoparticles used for reporting a color change is 100 pL of 3.4 nM.
- the detection limit is tunable by changing a value of m such that increasing the value of m improves the detection limit of the protease.
- one of the physicochemical properties is interfacial affinity
- the modular peptide is an affinity-switchable divalent peptide having a general formula (Asp) «-(AA) x -(Cys) m , and being synthesized via a Fmoc- SPPS method, an (Asp),, block of the modular peptide including at least Asp units and a (Cys) m block of the modular peptide including at least Cys units.
- the peptide is DDDTSAVLQSGFACGAGC.
- the modular peptide combines with gold nanoparticle modified with a citrate or tris(2-carboxyethyl)phosphine (TCEP) ligand.
- TCEP tris(2-carboxyethyl)phosphine
- the modular peptide is an amphiphilicity-switchable peptide having the general formula (Asp) «-(AA) x -(Phe-Phe-Pro) m -Cys, synthesized via a Fmoc-SPPS method, an (Asp),, block of the modular peptide including at least Asp units.
- n >2, and m > 1.
- the peptide combines with gold nanoparticles modified with a citrate or a diphenylphosphinobenzene sulfonate (DPPS) ligand.
- DPPS diphenylphosphinobenzene sulfonate
- the peptide is DDDTSAVLQSGFRCGRGC.
- a method for screening a drug inhibitor includes: incubating drug molecules of various concentrations with a protease of interest; performing colorimetric assays based on a mixture of a peptide and gold nanoparticles in a multiple well plate; measuring an absorbance ratio or color change for an indication of drug potency; and wherein the peptide is selected from the group including (i) a charge-switchable zwitterionic peptide having a general formula (Asp/Glu) ceremoni-(A A) Y -(Arg/Lys), jail, and being synthesized via a Fmoc-SPPS method, wherein an (Arg/Lys) m block of the peptide includes at least Arg and Lys units and an (Asp/Glu)horizon block of the charge-switchable zwitterionic peptide includes at least Asp and/or Glu units, (ii) an affinity-switchable divalent peptid
- the gold nanoparticles are stable with the drug molecules at testing concentrations.
- a diagnostic test strip includes: an absorbent pad on which a peptide is loaded and fixed; a blister pack having a gold nanoparticle dispersion sealed therein such that upon release from the blister pack the gold nanoparticle dispersion is fluidically communicated onto the absorbent pad, a peptide being selected such that a selected protease concentrated and incubated on the absorbent pad causes a visual indicator to indicate a presence of the selected protease; at least one carrier layer for supporting the absorbent pad and the blister pack; and wherein the peptide is selected from the group including (i) a charge-switchable zwitterionic peptide having a general formula (Asp/Glu) «-(AA)x-(Arg/Lys) m , and being synthesized via a Fmoc-SPPS method, wherein an (Arg/Lys) m block of the peptide includes at least Arg and Lys units and an (A
- the carrier layer includes an adhesive layer, the diagnostic test strip being configured to be removably securable to a face mask using the adhesive layer such that the selected protease concentrates on the absorbent pad from exhaled breadth condensate or saliva from a wearer of the face mask.
- the selected protease is M pro .
- a method of making a diagnostic test strip includes: loading and fixing a peptide on an absorbent pad; sealing a gold nanoparticle dispersion in a blister pack; arranging the blister pack to cooperate with the absorbent pad so that upon release from the blister pack the gold nanoparticle dispersion is fluidically communicated onto the absorbent pad, a peptide being selected such that a selected protease concentrated and incubated on the absorbent pad causes a visual indicator to indicate a presence of the selected protease; securing the absorbent pad and the blister pack on at least one carrier layer; and wherein the peptide is selected from the group including (i) a charge-switchable zwitterionic peptide having a general formula (Asp/Glu) «-(AA) x -(Arg/Lys) m , and being synthesized via a Fmoc-SPPS method, wherein an (Arg/Lys) m block of the
- the selected protease is M pro .
- a method of diagnostic testing includes: releasing a gold nanoparticle dispersion onto an absorbent pad loaded with a peptide after protease-containing patient samples have been concentrated and incubated on the absorbent pad; after releasing the gold nanoparticle dispersion, determining if a selected protease is present based on a change in visual appearance of the absorbent pad and wherein the peptide is selected from the group including (i) a charge-switchable zwitterionic peptide having a general formula (Asp/Glu) «-(AA)x-(Arg/Lys) m , and being synthesized via a Fmoc-SPPS method, wherein an (Arg/Lys) m block of the peptide includes at least Arg and Lys units and an (Asp/Glu),, block of the charge-switchable zwitterionic peptide includes at least Asp and/or Glu units, (i)
- the gold nanoparticle dispersion is stored in a blister pack, the blister pack and the absorbent pad being incorporated in a test strip.
- the method further includes applying the test strip onto a face mask such that the protease-containing patient samples are obtained from exhaled breadth condensate or saliva of a wearer of the face mask.
- the selected protease is M pro .
- a continuous poll by YouGov showed that the use of face coverings in public by Americans was 75-80% in early 2021. Face coverings naturally accumulate the exhaled saliva and other aerosolized droplets from the oral cavity. Accordingly, face coverings could offer both protective and diagnostic features when repurposed with analytical capabilities. That is, the exhaled respiratory droplets harbor diagnostic biomarkers (e.g., metabolites, hormones, enzymes, and toxins) that can be harvested from the face coverings and then used to test the wearer’s health status. For example, pathogenic nucleic acid and viral protease antibodies have recently been found in saliva droplets collected from COVID- 19-positive patients.
- diagnostic biomarkers e.g., metabolites, hormones, enzymes, and toxins
- a face covering is equipped with a sensing test strip that contains one or more reagents for detecting aerosolized salivary biomarkers that are collected by the face covering.
- FIG. 1 shows a schematic diagram of such a face covering.
- the face covering 10 may be of any type that includes a body 12 having one or more layers of material that are shaped to covers at least the mouth and nose of the wearer.
- the face covering 10 also includes a mechanism such as ear loops 14 or tie straps for affixing the face mask to the user’s face.
- Examples of such face coverings include cloth masks, surgical masks, neck gaitors and N95 respirator masks, KN95 respirator masks, FFP2 respirator masks or other face coverings that may or may not conform to an establish standard.
- the body 12 of such masks may include one or more layers (e.g., a tri-layer structure) of material.
- the sensing test strip 16 may be secured to the body 12 of the mask in a manner that allows it to be easily attached and removed.
- the backing layer may have a removable liner that exposes an adhesive layer that allows the test strip to be attached to a wide variety of different face mask types.
- the biomarker will be one or more proteases that are specific to SARs viruses and which are an enzymatic byproduct of a SARS virus infection.
- the protease that is detected to indicate a SARS infection may be the M pro (also known as 3CL pro ) and/or papain-like proteases (PLP).
- M pro also known as 3CL pro
- PBP papain-like proteases
- M pro Based on its critical function in the SARS viral life cycle, M pro has long been a target for antiviral drug development. Inhibitors targeting the M pro active site are designed to mimic the natural protease substrate. Importantly, M pro assays are likely to be SARS-specific since there are no known human proteases with similar substrate specificity as M pro .
- detection of M pro may be complemented with detection of a papain-like protease (PLP) to increase the specificity of the testing.
- PLP is another protease that is encoded by the viral genome and is specifically expressed during coronavirus infection. Similar to M pro , PLP is co-translated with viral replicase proteins as a single polypeptide. PLP is responsible for proteolytic processing of three non- structural proteins (nsps 1-3) located on the N-terminus of the replicase polyprotein. In infected cells, nsps 1-3 colocalize with sites of viral RNA replication. Additionally, nsp 1 has been shown to inhibit host mRNA translation.
- PLP In addition to playing a critical role in processing of the viral replicase, PLP also plays a role in inhibiting the innate immune response. PLP is known to inhibit multiple aspects of the anti-viral interferon pathway in host cells. Thus, PLP is a potential target for anti-viral drug and probe development. In these embodiments simultaneous detection of M pro and PLP will offer a selective means of detecting SARS-CoV2 infection.
- the sensing test strip that detects the proteases may employ colorimetric reagents that change color only in the presence of the target protease or proteases.
- the colorimetric reagent may be based on gold nanoparticles (AuNPs). Solutions of gold nanoparticles are normally red. However, when the particles become very close to each other, the solution becomes blue/purple. A suitable peptide can be used to cause electrostatic interactions that ensure that this color change occurs only in the presence of the M pro protease.
- the peptide that is selected may be a modular zwitterionic peptide having the general formula (Asp) n “(AA) x -(Arg)m, with n>m. These peptides have been found to induce AuNP aggregation in response to proteases.
- the zwitterionic peptide has both positive and negative building blocks. After M pro cleavage of the peptide, the positive-charged segments attract negatively-charged gold nanoparticles, thereby yielding a color change.
- FIG. 2 shows an exploded view of one particular embodiment of a sensing test strip that may be secured to a face mask in a removable manner.
- the test strip employs a multilayer structure that includes a cover layer 110 having one or more vents 102 to allow transfer of breath, saliva and aerosols to an underlying absorbent pad 112 that serves to concentrate the proteases.
- a spacer layer 114 has precut channels in which the absorbent pad 112 is placed.
- a reagent housing 115 such as a blister pack, which stores the reagent in the liquid phase, is also placed in a precut opening of the spacer layer 114 so that upon release of the reagent (e.g., upon opening of the blister pack by compressing it) it flows into the absorbent pad 112 to interact with the captured biomarkers.
- the spacer 114, along with the pad 112 and reagent housing 115 is placed onto a support layer 116 that has vents 117 that are aligned with the vents 101 in the cover layer 110.
- Adhesive on the lower surface of the cover layer 110 and the upper surface of the support layer 116 can be used to secure the spacer 115 to both the cover layer 110 and the support layer 116.
- the bottom surface of the support layer 116 may also be coated with an adhesive that is covered with a release liner that the user removes when the sensing test strip is to be secured to the face mask.
- the absorbent pad 112 may include two flow channels or paths 112A and 112B, one of which serves as a positive control that is treated with recombinant protease biomarkers. The other flow channel or path will remain untreated and exposed to air.
- the cover layer 110 is transparent to allow for visual analysis of both flow channels after the reagent has been released from the reagent housing 115.
- the sensing test strip may be about 4cm in length and 2 cm wide and the reagent housing 115 may be a blister pack that contains about 500 pL of the reagent. More generally, the size, shape and overall structure and configuration of the sensing test strip may be application specific and is not limited to the example described herein.
- FIG. 3 A shows the overall structure of the sensing test strip that was used, which is largely the same as the test strip described above in connection with Fig. 2.
- the vents on the front cover Pl and the bottom adhesive layer P6 had an area of about 0.72 cm 2 .
- Figs. 3C-3F denotes the level of salivary amylase, which is proportional to the amount of saliva droplets accumulated on the masks.
- the amylase level on the mask materials was also included as a control (the most right in each box plot in Figs. 3C-3F).
- the test strip placed inside the face coverings still captured enough amylase for detection.
- the determined amylase levels were 1,166 U/L for the strip placed inside neck gaiter, 533 U/L for that of the cloth mask, and 2,200 U/L for the strip inside surgical mask.
- the test strip had low efficiency of saliva collection (300 U/L). This could arise from the large breathing room dispersing the amylase at a low amount per area.
- FIG. 4A shows that the sensing test strip that was used was based on the design shown in Fig. 3 A, except that an extra layer of forensic press paper P4 was inserted underneath the polyester pad P5.
- the press paper P4 acts as a blue color agent.
- the inset in FIG. 4A shows the detection mechanism.
- the blister pack P5 that serves as the reagent storage was loaded with deionized water.
- Fig. 4B shows white-light images of the amylase-sensing test strip before and after release of the water.
- the test line on the pad shifted its color from white to blue (represented by the darker regions in Fig. 4B), which can be viewed from both the front and back sides.
- the sealed control line without exposing to any oral droplets retained its original color in white.
- Colorimetric sensors based on gold nanoparticles are popular because of their unique photophysical properties including a surface plasmon resonance (SPR) band with a high molar absorption coefficient. Aggregation enhances the near-field interactions of proximal nanoparticles and leads to a bathochromic shift in the SPR band; this in turn leads to a pronounced color change.
- Strategies for plasmonic coupling can be grouped into chemical linking and physical factors. For example, dithiothreitol is a simple molecule to cluster AuNPs due to the strong thiol-to-gold bond. Nanoplasmonic sensing based on this covalent bond have been reported for thrombin, trypsin, furin, etc.
- controlled physical factors e.g., ionic strength, solvent polarity, and ligand hydrophilicity
- nanoparticles primarily stabilized through electrostatic double layer repulsions tend to aggregate via the addition of excess salt due to charge screening.
- Tuning non-specific electrostatic interactions has also led to several simple and rapid colorimetric assays for heavy metal ions.
- these electrostatic processes could be repurposed for sensing other targets (here, proteases) by rationally designing the electrophoretic property of a specific substrate where the selectivity is based on molecular recognition between the enzyme and the substrate.
- the modular zwitterionic peptide has oppositely charged termini, /. ⁇ ., (Asp),, and (Arg) m , flanking a specific M pro cleavage site (AA) X . Because n > m, the intact peptide carries a net neutral charge. Accordingly, the interactions between the intact peptide and sulfonated ligand-coated AuNPs are minimized given the negatively charged sulfonate groups. Proteolytic segmentation of the peptide would switch the electrophoretic property of the corresponding fragment, thus triggering the collapse of gold suspension via electrostatic attractions. This induces a visible color change.
- FIG. 5A schematically shows the intact peptide and its proteolytic fragments with distinct physicochemical properties (e.g., net charge, interfacial affinity) to modulate the dispersity of a gold suspension.
- Fig. 5A shows a schematic illustration of the aggregation of bis(/?-sulfonatophenyl)phenylphosphine-coated gold nanoparticles (BSPP-AuNPs) caused by the proteolytic hydrolysis of the intact peptide, where the net charge of intact peptide and its fragment is reversed.
- the cartoon designates proteases, /. ⁇ ., M pro ; the tandem hexagon represents a modular zwitterionic peptide with a M pro recognition site.
- Fig. 5B shows that the colorimetric test could be coupled with face coverings with a lateral flow strip to indicate CO VID infection in-situ (top).
- Fig. 5C show TEM images of the dispersed BSPP-AuNPs (top) and proteolysis-induced gold aggregates [bottom, (i)].
- the flocculated AuNPs can be redispersed using ionic surfactant additives, such as sodium dodecyl sulfate [SDS, (ii)], due to the restored electrostatic repulsion.
- ionic surfactant additives such as sodium dodecyl sulfate [SDS, (ii)
- the zwitterionic peptides were rationally designed to encompass arranging three functional domains in a linear N-to-C-terminal configuration (Fig. 6A), including: (i) an N-terminal charge shielding domain made of repeating aspartic acid (Asp, D) for neutralizing the charge of the intact peptide, /. ⁇ ., part iii; (ii) an exposed protease-recognition sequence; and (iii) a C-terminal aggregating site for clustering gold colloids.
- the aggregating domain (part iii) has a repeating arginine (Arg, R) and a cysteine (Cys, C) bridge with high affinity to AuNPs via electrostatic and coordinating interactions.
- the first is little or no attraction between the intact peptide and AuNPs. This is achieved by matching the charge state of intact peptide to that of particle surfaces, which typically originates from the terminal groups of the surface ligand.
- Second, peptide fragmentation by the protease exposes the aggregating domain that favors tight self-assembly of particles. This modular design is also intended to obviate the need for major revisions when targeting a new protease.
- Fig. 6B shows HPLC and ESI-MS data showing that M pro selectively cleaves the ZY7 peptide at the C-terminus of glutamine (Q). The peak with * is the product. See also Table 1 for additional information concerning the ZY7 and other peptides.
- the functional peptide is used under label-free conditions with AuNPs as a readout to detect the presence of M pro . This is advantageous because surface labelling techniques often require precise control over the bio-nano functionalization and may involve frequently unanticipated particle aggregation.
- BSPP bis( - sulfonatophenyl)phenylphosphine
- BSPP- capped AuNPs have a compact ligand layer and thus favor tight interparticle interactions, e.g., the hydrodynamic diameter (DH) is 19.1 nm with a PDI of 0.10.
- DH hydrodynamic diameter
- M pro peptidase as a model example to test our label-free peptide and BSPP -AuNPs. M pro regulates viral replication and transcription and is therefore an attractive therapeutic and diagnostic target for SARS-CoV-2.
- 6C shows the color change (in gray scale) as a function of the peptide concentration and time.
- ZY7 fragments (6-50 pM)
- BSPP-AuNPs coexist with the ZY7 parent peptide with no change of the color.
- Fig. 5C shows TEM images in Fig. 5C and hydrodynamic diameter (DH) by dynamic light scattering (DLS) clearly indicate the formation of AuNP aggregates in the presence of ZY7 fragments.
- Fig. 6D shows DLS profiles of BSPP-AuNPs (3.8 nM, 100 pL) incubated with increasing concentrations of ZY7 parent peptide (darker circles) and ZY7 fragments (lighter circles).
- the time- lapsed absorbance profiles of AuNPs with ZY7 parent peptide remain essentially the same indicating a preserved dispersity.
- the ZY7 fragment led to a sizable decrease in the SPR at 520 nm commensurate with a noticeably raising band at 600 nm. This is a clear indication of AuNP aggregation and color change.
- the above mechanism favors electrostatic-induced flocculation of AuNPs and was further corroborated by a reversible colorimetric experiment, employing several different surfactants.
- ionic surfactants such as cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) recovered colloidal AuNPs from the aggregates. Excess anionic SDS scavenges the positively charged peptide fragments, thus restoring the electrostatic double layer repulsions between particles.
- Fig. 7B shows the time progression of absorbance ratio in the enzyme assay, where a fixed amount of ZY7 substrate (50 pM) is incubated with increasing concentrations M pro (0-200 nM).
- the test media is exhaled breath condensate (EBC).
- EBC exhaled breath condensate
- the data points were read every 1 min for 1 hour. The trend clearly indicates that higher amount of M pro led to more particle aggregations or rapid color change and vice versa.
- BSPP-AuNPs were stable in the presence of M pro alone and in all tested buffers.
- Fig. 7C shows the ratio of Abseoo/Abs52o as a function of M pro concentration.
- the LoD for M pro was determined to be: 27.7 nM in Tris buffer (TB), 33.4 nM in EBC matrix, and 68.4 nM in 50% saliva.
- the method for LoD calculation is reported in previous literature. This suggests that our sensor is best performing in TB and EBC media, while other complex matrices such as saliva would yield sensitivity in about 3-fold lower or more. No aggregation occurred in human plasma presumably negative proteins that scavenge the positively charged peptide fragments.
- MS7 peptide we substituted the indispensable glutamine (Q) at Pl site with its analogue, glutamic acid (E), and therefore no specific recognition by M pro , circumventing the formation of aggregating fragments; see Table 1.
- the colorimetric detection can be carried out in a one-pot assay. The enzymatic cleavage and particle aggregation took place simultaneously in-situ, albeit a longer readout time was needed ( ⁇ 1 h). The LoD of the one-pot assay for M pro is about 40.9 nM.
- FIGs. 8 A, 8B and 8C show the operation window of M pro sensors based on NR10, OR8, and YR9 peptide, which contains 0, 1, and 4 arginine residues in its aggregating sequence, respectively.
- the aggregating sequences containing zero or single guanidine side chain produce no optical signal change.
- These non-aggregating systems have low ionic valence and attenuated electrostatic interactions according to the Schulze-Hardy rule.
- Fig. 8D shows the M pro LoD of sensors based on four peptides of varying number of arginine: no LoD observed for NR10 and OR8 substrate; 27.7 and 3.4 nM is determined for YR9 and ZY7 substrate, respectively.
- Fig. 8E shows the results of assaying an increasing molarity of GC376 (i.e., 0-1 pM) in the presence of constant amount of M pro (100 nM) and ZY7 substrate (50 pM). Note that the inhibitor itself does not affect the particle dispersity; see the control line in Fig. 8E. As anticipated, the aggregation kinetics were largely retarded due to low activities of pre-mixed M pro with the inhibitor.
- Fig. 8G shows sensor activation by other mammalian proteins (100 nM), including bovine serum albumin (BSA), hemoglobin, a-amylase (100 U/mL), thrombin, and trypsin. Assay with and without M pro is included as positive and negative control.
- BSA bovine serum albumin
- a-amylase z.e., digests a-l,4-glucosidic bond in starch
- thrombin z.e., cleaves Arg-Gly bond in fibrinogen
- trypsin z.e., cleaves at C-terminus of Arg or Lys
- Fig. 8G shows sensor activation by other mammalian proteins (100 nM), including bovine serum albumin (BSA), hemoglobin, a-amylase (100 U/mL), thrombin, and trypsin.
- Assay with and without M pro is included as positive and negative control.
- ESI-MS data confirmed that ZY7 substrate was recognized by trypsin at the C-terminal of arginine at P4’ site, exposing a short CGRGC fragment that causes the AuNP aggregation.
- LoD for MP ro is 27.7 and 114.4 nM for ZY7 and DS12, respectively; LoD for trypsin is 9.7 and 30.3 nM for ZY7 and DS 12, respectively.).
- no background activation was observed for the sensor based on DS 12 in saliva media.
- the LoD for M pro using DS 12 substrate showed an approximately 3-fold reduction comparing to that of ZY7 substrate (Table 1).
- the loss in sensitivity is in alignment with previous reports of a 4-fold reduction in M pro efficiency on alteration of the P4’ arginine.
- peptide/ AuNP -based protease detection resides in its simplicity where no complex bioconjugation techniques or readout instrumentation are required. This allows for the large-scale implementation of qualitive diagnostics for protease markers. As previously discussed, tests like these can be easily affixed to routinely used face coverings, turning them into easy-to-use diagnostic kits. As also described earlier, the detection technique described herein can be adapted onto a flowcell inspired M pro sensing strip of the type described above in connection with Fig. 2 and further shown in Fig. 9A and Fig.
- FIG. 9B which shows a white light image of the assembled sensing test strip, equipped with a BSPP-AuNP dispersion in the blister pack, ZY7 peptide on the test lane (left, dash box), and YF15 peptide on the positivecontrol lane.
- the test strip consisted of two pad lanes: (i) A test lane decorated with 50 pM of ZY7 peptide, and (ii) a positive control lane loaded with 50 pM of peptide fragment (YF15). This peptide fragment is the most potent aggregating domain from all the peptides listed in Table 1.
- 60 pL of BSPP-AuNPs was housed in the blister pack and used for readout or color agent (Fig. 9B). In principle, one pink and one purple lane would signify a COVID-negative test, while two purple lanes indicate a positive test for the presence of M pro .
- Fig. 9C shows various concentrations of M pro in EBC (0-100 nM, with respect to 30 pL volume) tested for 10 min.
- the test lanes incubated with M pro more than 40 nM started to appear as purple color, indicating positive test results.
- Figs. 9E and 9F show SEM images of non-aggregating AuNPs on the red lane and clustered AuNPs on the purple lane, respectively.
- the strip/pad affixed on a face covering would collect the respiratory droplets that harbor M pro biomarker (for 8 h or a normal workday). Meanwhile, on the absorbent pad the concentrated M pro proteases start to cleave the pre-loaded ZY7 peptide. At the end of the day, the BSPP-AuNPs in the blister pack are released and moved to the absorbent pad via capillary action to interact with the peptides for 10 min readout.
- a proof-of-concept experiment we show here ten EBC samples collected from the COVID-negative volunteers (/. ⁇ ., verified by PCR test) and showed no false positives (see Fig.
- FIG. 9E shows an SEM image of non-aggregating AuNPs on the red lane and
Abstract
A colorimetric assay uses a stimuli-responsive peptide for detecting an enzyme. The assay includes a modular peptide having physicochemical properties that change in response to a presence of an enzyme of interest such as a protease. The assay also includes a color reagent indicating a presence and quantitative level of the enzyme of interest when treated with the modular peptide via a visual readout. The quantitative level of modular peptide activation is related to a quantity of the enzyme of interest such that the colorimetric assay is applicable for purposes of inhibitor screening and diagnostic testing.
Description
A FACE COVERING HAVING A TEST STRIP FOR COLORIMETRIC MONITORING OF PROTEASES AND METHODS OF DETECTING PROTEASES
GOVERNMENT FUNDING
[0001] This invention was made with government support under DE031114 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND
[0002] The COVID-19 pandemic is caused by the SARS-CoV-2 virus transmitted via aerosols or droplets expelled by speaking, breathing, or coughing.1,2 Non- pharmacological interventions (e.g., mask-wearing and diagnostic testing) have been crucial in preventing the ongoing spread of SARS-CoV-2 prior to vaccine deployments. However, Vulnerable populations do not just need testing — they need surveillance. Vulnerable populations such as residents (and caregivers) in group care homes, prisons, dialysis clinics, etc. are very susceptible to SARS-CoV-2 infection. Therefore, they should be tested frequently to quickly identify and isolate infected persons while notifying their contacts. An ideal test could be performed at the point- of-care by the person to be tested or their caregiver — it would be simple, affordable, reliable, and accurate.
[0003] PCR and serology are the current gold standards to diagnose SARS-CoV-2 infection but have critical limitations. Serology only indicates prior exposure with few details on current viral activity. PCR requires a complex laboratory infrastructure, trained personnel, and multi-step sample preparation. Thus, widespread surveillance efforts will continue to suffer until these testing shortfalls are resolved.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] FIG. 1 shows a schematic diagram of one example of a face covering that incorporates a sensing test strip.
[0005] FIG. 2 shows an exploded view of one example of the sensing test strip shown in Fig. 1.
[0006] FIG. 3 A shows another exploded view of the sensing test strip shown in FIG. 2; FIG. 3B is a white-light image of a test strip affixed to the outside of a neck gaiter and another test strip offset parallel inside the mask; and Figs. 3C, 3D, 3E and 3F show the measured amylase level on the outside and inside test strips attached to different face coverings for a neck gaiter, for a cloth mask, a surgical mask, and a N95 respirator, respectively.
[0007] Fig. 4A shows an example of an amylase-sensing test strip similar to that shown in Figs. 2 and 3 A but which includes an extra layer of forensic press paper underneath the polyester pad to act as a blue color agent; and Fig. 4B shows whitelight images of the amylase-sensing test strip before and after release of water.
[0008] Fig. 5 A shows a schematic illustration of the aggregation of bis(/?- sulfonatophenyl)phenylphosphine-coated gold nanoparticles (BSPP-AuNPs) caused by the proteolytic hydrolysis of the intact peptide, where the net charge of intact peptide and its fragment is reversed; Fig. 5B shows that a colorimetric test coupled with face coverings having a lateral flow strip to indicate COVID infection in-situ (top); and Fig. 5C shows TEM images of the dispersed BSPP-AuNPs (top) and proteolysis-induced gold aggregates [bottom, (i)] and flocculated AuNPs redispersed using ionic surfactant additives [(ii)], due to the restored electrostatic repulsion.
[0009] Fig. 6A shows a representative peptide ZY7; Fig. 6B shows HPLC and ESIMS data showing that Mpro selectively cleaves the ZY7 peptide at the C-terminus of glutamine (Q); Fig. 6C shows the color change (in gray scale) as a function of peptide concentration and time; Fig. 6D shows DLS profiles of BSPP-AuNPs (3.8 nM, 100 pL) incubated with increasing concentrations of ZY7 parent peptide (darker circles) and ZY7 fragments (lighter circles); Fig. 6E shows zeta potential measurements of AuNPs (3.8 nM, 100 pL) incubated with increasing concentrations of ZY7 parent peptide (darker circles) and ZY7 fragments (lighter circles); and Figs. 6F-6G show the time progression of optical absorption of AuNPs (3.8 nM, 50 pL) when incubated with ZY7 parent peptide and its fragments (c = 6.0 pM).
[0010] Fig. 7A shows the ratiometric signal (Abseoo/Abs52o) collected from BSPP- AuNPs (3.8 nM, 100 pL) incubated with various amount of ZY7 parent and fragments; Fig. 7B shows the time progression of absorbance ratio in the enzyme assay, where a fixed amount of ZY7 substrate (50 pM) is incubated with increasing
concentrations Mpro (0-200 nM); Fig. 7C shows the ratio of Abseoo/Abs52o as a function of Mpro concentration; and Fig. 7D, which shows the absorbance ratio as a function of Mpro concentration in three biofluids, where MS7 (control) substrate (50 pM) is used.
[0011] FIGs. 8 A, 8B and 8C show the operation window of Mpro sensors based on NR.10, OR8, and YR9 peptide, which contains 0, 1, and 4 arginine residues in its aggregating sequence, respectively; Fig. 8D shows the Mpro LoD of sensors based on four peptides of varying number of arginine; Fig. 8E shows the results of assaying an increasing molarity of GC376 (/.<?., 0-1 pM) in the presence of constant amount of Mpro (100 nM) and ZY7 substrate (50 pM); Fig. 8F shows a typical inhibition titration curve fitted with the Morrison equation for the competitive inhibitor, GC376, where the inset shows the chemical structure of GC376 inhibitor; Fig. 8G shows sensor activation by other mammalian proteins (100 nM), including bovine serum albumin (BSA), hemoglobin, a-amylase (100 U/mL), thrombin, and trypsin; and Fig. 8H shows the response of sensors based on ZY7 and DS 12 substrate to Mpro or trypsin in Tris buffer.
[0012] FIG. 9A show an illustration of the sensing test strip on a face mask and an exploded view of the sensing test strip; FIG. 9B shows a white light image of the assembled sensing test strip, equipped with a BSPP-AuNP dispersion in the blister pack, ZY7 peptide on the test lane (left, dash box), and YF15 peptide on the positivecontrol lane; Fig. 9C shows various concentrations of Mpro in EBC (0-100 nM, with respect to 30 pL volume) tested for 10 min; Fig. 9D shows strip testing of the Mpro marker on COVID-negative participants (NP#, 77=10); FIG. 9E shows an SEM image of non-aggregating AuNPs on the red lane and FIG. 9F shows an SEM image of clusters AuNPs on the purple lane; and Fig. 9G shows sensor testing on aqueous EBC matrices collected from COVID-negative subjects (77=10).
SUMMARY
[0013] In accordance with one aspect of the subject matter described herein, a colorimetric assay uses a stimuli-responsive peptide for detecting an enzyme. The assay includes a modular peptide having physicochemical properties that change in response to a presence of an enzyme of interest. The assay also includes a color reagent indicating a presence and quantitative level of the enzyme of interest when treated with the modular peptide via a visual readout. The quantitative level of
modular peptide activation is related to a quantity of the enzyme of interest such that the colorimetric assay is applicable for purposes of inhibitor screening and diagnostic testing.
[0014] In accordance with a particular aspect, one of the physicochemical properties is charge potential, the modular peptide being a charge-switchable zwitterionic peptide having a general formula (Asp/Glu)«-(AA)x-(Arg/Lys)m, and being synthesized via a Fmoc-SPPS method, wherein an (Arg/Lys)m block of the modular peptide includes at least Arg and Lys units and an (Asp/Glu)„ block of the modular peptide includes at least Asp and/or Glu units.
In accordance with a particular aspect, the color reagent includes negatively -charged- ligand functionalized plasmonic nanoparticles.
[0015] In accordance with a particular aspect, when intact, the peptide is in a net neutral or negative charge state that has minimal interactions with the functionalized plasmonic nanoparticles, with n > m > 2.
In accordance with a particular aspect, -(AA)r- is an amino acid sequence of varying length comprising natural and synthetic amino acids that is responsive to the enzyme of interest, the enzyme of interest being selected from the group consisting of a viral protease, a mammalian protease, or a combinations thereof
[0016] In accordance with a particular aspect, installed ligands are phenyl phosphine derivatives selected from the group consisting of: diphenylphosphinobenzene sulfonate (DPPS), bis(p-sulfonatophenyl)phenylphosphine (BSPP), triphenylphosphine-3,3,3-trisulfonate (TPPTS), and combinations of the above.
[0017] In accordance with a particular aspect, a charged ligand is installed on plasmonic nanoparticles by a ligand exchange or phase transfer reaction.
[0018] In accordance with a particular aspect, plasmonic particles of the negatively- charged-ligand functionalized plasmonic nanoparticles are prepared from citrate- stabilized spherical gold nanoparticles by a Turkevich method.
[0019] In accordance with a particular aspect, a negative charge on the peptide arises from aspartic acid, glutamic acid, or a combination of thereof and a positive charge arises from arginine, lysine, or a combination thereof.
[0020] In accordance with a particular aspect, the charge-switchable zwitterionic peptide is used with gold nanoparticles under label free conditions.
In accordance with a particular aspect, a mass concentration for ligand exchange is 0.5 to 2 mg of ligand per mL of nanoparticle volume.
[0021] In accordance with a particular aspect, the enzyme of interest is a protease, a peptide fragment arising from protease breakdown being a positively-charged species that electrostatically interacts with gold nanoparticles.
[0022] In accordance with a particular aspect, an electrostatic attraction of the gold nanoparticle induces a plasmonic coupling and color changes.
[0023] In accordance with a particular aspect, a color change arising from nanoparticle aggregation occurs either after introducing the protease pre-mixed with the modular peptide to a dispersion of the gold nanoparticle or after introducing the protease to the modular peptide premixed with the gold nanoparticles.
[0024] In accordance with a particular aspect, incubation of the protease with the modular peptide occurs at 37 °C for 0.5 to 3 hours, with a molar ratio of [peptide]: [protease] varying from 250 to 100,000.
[0025] In accordance with a particular aspect, color changes at 10 min are recorded by eye or using a smart phone, an optical absorption spectrometer, or both.
[0026] In accordance with a particular aspect, the gold nanoparticles are stable in buffers for no less than 24 hours.
[0027] In accordance with a particular aspect, a sample to be analyzed contains biological samples.
[0028] In accordance with a particular aspect, the biological samples are selected from the group including exhaled breath condensate, saliva, human plasma, urine and gingival crevicular fluid.
[0029] In accordance with a particular aspect, the biological samples further include buffers.
[0030] In accordance with a particular aspect, the buffers include dithiothreitol.
[0031] In accordance with a particular aspect, a detection limit of the protease is at a first molar ratio of [peptide]: [protease] that shows an observable color change.
[0032] In accordance with a particular aspect, an amount of gold nanoparticles used for reporting a color change is 100 pL of 3.4 nM.
[0033] In accordance with a particular aspect, the detection limit is tunable by changing a value of m such that increasing the value of m improves the detection limit of the protease.
[0034] In accordance with a particular aspect, one of the physicochemical properties is interfacial affinity, the modular peptide is an affinity-switchable divalent peptide
having a general formula (Asp)«-(AA)x-(Cys)m, and being synthesized via a Fmoc- SPPS method, an (Asp),, block of the modular peptide including at least Asp units and a (Cys)m block of the modular peptide including at least Cys units.
[0035] In accordance with one particular aspect, the peptide is DDDTSAVLQSGFACGAGC.
[0036] In accordance with a particular aspect, n >2, and m = 2.
[0037] In accordance with a particular aspect, the modular peptide combines with gold nanoparticle modified with a citrate or tris(2-carboxyethyl)phosphine (TCEP) ligand.
[0038] In accordance with a particular aspect, one the physicochemical properties is amphiphilicity, the modular peptide is an amphiphilicity-switchable peptide having the general formula (Asp)«-(AA)x-(Phe-Phe-Pro)m-Cys, synthesized via a Fmoc-SPPS method, an (Asp),, block of the modular peptide including at least Asp units.
[0039] In accordance with a particular aspect, n >2, and m > 1.
[0040] In accordance with a particular aspect, the peptide combines with gold nanoparticles modified with a citrate or a diphenylphosphinobenzene sulfonate (DPPS) ligand.
[0041] In accordance with a particular aspect, the peptide is DDDTSAVLQSGFRCGRGC.
[0042] In accordance with another aspect, a method is presented for screening a drug inhibitor. The method includes: incubating drug molecules of various concentrations with a protease of interest; performing colorimetric assays based on a mixture of a peptide and gold nanoparticles in a multiple well plate; measuring an absorbance ratio or color change for an indication of drug potency; and wherein the peptide is selected from the group including (i) a charge-switchable zwitterionic peptide having a general formula (Asp/Glu)„-(A A)Y-(Arg/Lys),„, and being synthesized via a Fmoc-SPPS method, wherein an (Arg/Lys)m block of the peptide includes at least Arg and Lys units and an (Asp/Glu)„ block of the charge-switchable zwitterionic peptide includes at least Asp and/or Glu units, (ii) an affinity-switchable divalent peptide having a general formula (Asp)«-(AA)x-(Cys)m, and being synthesized via a Fmoc-SPPS method, an (Asp),, block of the affinity-switchable divalent peptide including at least Asp units and a (Cys)m block of the affinity-switchable divalent peptide including at least Cys units, and (iii) an amphiphilicity-switchable peptide having a general
formula (Asp)«-(A A)Y-(Phe-Phe-Pro)m-Cys, synthesized via a Fmoc-SPPS method, an ( Asp),, block of the amphiphilicity-switchable peptide including at least Asp units. [0043] In accordance with a particular aspect, the drug molecules are incubated with the protease of interest for 10 to 30 min prior to incubation with the peptide.
[0044] In accordance with a particular aspect, the gold nanoparticles are stable with the drug molecules at testing concentrations.
[0045] In accordance with another aspect, a diagnostic test strip is provided. The diagnostic test strip includes: an absorbent pad on which a peptide is loaded and fixed; a blister pack having a gold nanoparticle dispersion sealed therein such that upon release from the blister pack the gold nanoparticle dispersion is fluidically communicated onto the absorbent pad, a peptide being selected such that a selected protease concentrated and incubated on the absorbent pad causes a visual indicator to indicate a presence of the selected protease; at least one carrier layer for supporting the absorbent pad and the blister pack; and wherein the peptide is selected from the group including (i) a charge-switchable zwitterionic peptide having a general formula (Asp/Glu)«-(AA)x-(Arg/Lys)m, and being synthesized via a Fmoc-SPPS method, wherein an (Arg/Lys)m block of the peptide includes at least Arg and Lys units and an (Asp/Glu)„ block of the charge-switchable zwitterionic peptide includes at least Asp and/or Glu units, (ii) an affinity-switchable divalent peptide having a general formula (Asp)«-(AA)x-(Cys)m, and being synthesized via a Fmoc-SPPS method, an (Asp),, block of the affinity-switchable divalent peptide including at least Asp units and a (Cys)m block of the affinity-switchable divalent peptide including at least Cys units, and (iii) an amphiphilicity-switchable peptide having a general formula (Asp)„-(AA)Y- (Phe-Phe-Pro)m-Cys, synthesized via a Fmoc-SPPS method, an (Asp),, block of the amphiphilicity-switchable peptide including at least Asp units.
[0046] In accordance with one particular aspect, the carrier layer includes an adhesive layer, the diagnostic test strip being configured to be removably securable to a face mask using the adhesive layer such that the selected protease concentrates on the absorbent pad from exhaled breadth condensate or saliva from a wearer of the face mask.
[0047] In accordance with one particular aspect, the selected protease is Mpro.
[0048] In accordance with another aspect, a method of making a diagnostic test strip is presented. The method includes: loading and fixing a peptide on an absorbent pad; sealing a gold nanoparticle dispersion in a blister pack; arranging the blister pack to
cooperate with the absorbent pad so that upon release from the blister pack the gold nanoparticle dispersion is fluidically communicated onto the absorbent pad, a peptide being selected such that a selected protease concentrated and incubated on the absorbent pad causes a visual indicator to indicate a presence of the selected protease; securing the absorbent pad and the blister pack on at least one carrier layer; and wherein the peptide is selected from the group including (i) a charge-switchable zwitterionic peptide having a general formula (Asp/Glu)«-(AA)x-(Arg/Lys)m, and being synthesized via a Fmoc-SPPS method, wherein an (Arg/Lys)m block of the peptide includes at least Arg and Lys units and an (Asp/Glu),, block of the charge- switchable zwitterionic peptide includes at least Asp and/or Glu units, (ii) an affinity- switchable divalent peptide having a general formula (Asp)«-(AA)x-(Cys)m, and being synthesized via a Fmoc-SPPS method, an (Asp),, block of the affinity-switchable divalent peptide including at least Asp units and a (Cys)m block of the affinity- switchable divalent peptide including at least Cys units, and (iii) an amphiphilicity- switchable peptide having a general formula (Asp)«-(AA)x-(Phe-Phe-Pro)m-Cys, synthesized via a Fmoc-SPPS method, an (Asp),, block of the amphiphilicity- switchable peptide including at least Asp units.
[0049] In accordance with one particular aspect, the selected protease is Mpro.
[0050] In accordance with another aspect, a method of diagnostic testing is provided. The method includes: releasing a gold nanoparticle dispersion onto an absorbent pad loaded with a peptide after protease-containing patient samples have been concentrated and incubated on the absorbent pad; after releasing the gold nanoparticle dispersion, determining if a selected protease is present based on a change in visual appearance of the absorbent pad and wherein the peptide is selected from the group including (i) a charge-switchable zwitterionic peptide having a general formula (Asp/Glu)«-(AA)x-(Arg/Lys)m, and being synthesized via a Fmoc-SPPS method, wherein an (Arg/Lys)m block of the peptide includes at least Arg and Lys units and an (Asp/Glu),, block of the charge-switchable zwitterionic peptide includes at least Asp and/or Glu units, (ii) an affinity-switchable divalent peptide having a general formula (Asp)«-(AA)x-(Cys)m, and being synthesized via a Fmoc-SPPS method, an (Asp),, block of the affinity-switchable divalent peptide including at least Asp units and a (Cys)m block of the affinity-switchable divalent peptide including at least Cys units, and (iii) an amphiphilicity-switchable peptide having a general formula (Asp)w-(AA)X-
(Phe-Phe-Pro)m-Cys, synthesized via a Fmoc-SPPS method, an (Asp),, block of the amphiphilicity-switchable peptide including at least Asp units.
[0051] In accordance with one particular aspect, the gold nanoparticle dispersion is stored in a blister pack, the blister pack and the absorbent pad being incorporated in a test strip.
[0052] In accordance with one particular aspect, the method further includes applying the test strip onto a face mask such that the protease-containing patient samples are obtained from exhaled breadth condensate or saliva of a wearer of the face mask.
[0053] In accordance with one particular aspect the selected protease is Mpro.
[0054] This Summary is provided to introduce a selection of concepts in a simplified form. The concepts are further described in the Detailed Description section.
Elements or steps other than those described in this Summary are possible, and no element or step is necessarily required. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended for use as an aid in determining the scope of the claimed subject matter. The claimed subject matter is not limited to implementations that solve any or all disadvantages noted in any part of this disclosure.
DETAILED DESCRIPTION
Introduction
[0055] A continuous poll by YouGov showed that the use of face coverings in public by Americans was 75-80% in early 2021. Face coverings naturally accumulate the exhaled saliva and other aerosolized droplets from the oral cavity. Accordingly, face coverings could offer both protective and diagnostic features when repurposed with analytical capabilities. That is, the exhaled respiratory droplets harbor diagnostic biomarkers (e.g., metabolites, hormones, enzymes, and toxins) that can be harvested from the face coverings and then used to test the wearer’s health status. For example, pathogenic nucleic acid and viral protease antibodies have recently been found in saliva droplets collected from COVID- 19-positive patients.
[0056] Experiments have been performed which demonstrate that conventional face coverings were effective at capturing aerosolized saliva droplets. These experiments used a colorimetric test to quantify a canonical salivary biomarker (i.e., a-amylase) and mapped the accumulation of respiratory droplets on four types of face coverings.
Selected results of these experiments will be presented below in the section entitled Proof of Concept.
[0057] In accordance with the subject matter disclosed herein, a face covering is equipped with a sensing test strip that contains one or more reagents for detecting aerosolized salivary biomarkers that are collected by the face covering. FIG. 1 shows a schematic diagram of such a face covering. The face covering 10 may be of any type that includes a body 12 having one or more layers of material that are shaped to covers at least the mouth and nose of the wearer. The face covering 10 also includes a mechanism such as ear loops 14 or tie straps for affixing the face mask to the user’s face. Examples of such face coverings include cloth masks, surgical masks, neck gaitors and N95 respirator masks, KN95 respirator masks, FFP2 respirator masks or other face coverings that may or may not conform to an establish standard. The body 12 of such masks may include one or more layers (e.g., a tri-layer structure) of material. The sensing test strip 16 may be secured to the body 12 of the mask in a manner that allows it to be easily attached and removed. For this purpose the backing layer may have a removable liner that exposes an adhesive layer that allows the test strip to be attached to a wide variety of different face mask types.
[0058] In some embodiments the biomarker will be one or more proteases that are specific to SARs viruses and which are an enzymatic byproduct of a SARS virus infection. In particular the protease that is detected to indicate a SARS infection (e.g., a SARS-CoV-2 infection) may be the Mpro (also known as 3CLpro) and/or papain-like proteases (PLP). The function of the Mpro protease in the viral life cycle is well understood. Upon viral entry into a host cell, Mpro is expressed together with non- structural viral proteins as a single polypeptide. The polypeptide undergoes selfcleavage at specific amino acid sequences leading to viral replication and transcription domains. Based on its critical function in the SARS viral life cycle, Mpro has long been a target for antiviral drug development. Inhibitors targeting the Mpro active site are designed to mimic the natural protease substrate. Importantly, Mpro assays are likely to be SARS-specific since there are no known human proteases with similar substrate specificity as Mpro.
[0059] In some embodiments detection of Mpro may be complemented with detection of a papain-like protease (PLP) to increase the specificity of the testing. PLP is another protease that is encoded by the viral genome and is specifically expressed during coronavirus infection. Similar to Mpro, PLP is co-translated with viral replicase
proteins as a single polypeptide. PLP is responsible for proteolytic processing of three non- structural proteins (nsps 1-3) located on the N-terminus of the replicase polyprotein. In infected cells, nsps 1-3 colocalize with sites of viral RNA replication. Additionally, nsp 1 has been shown to inhibit host mRNA translation. In addition to playing a critical role in processing of the viral replicase, PLP also plays a role in inhibiting the innate immune response. PLP is known to inhibit multiple aspects of the anti-viral interferon pathway in host cells. Thus, PLP is a potential target for anti-viral drug and probe development. In these embodiments simultaneous detection of Mpro and PLP will offer a selective means of detecting SARS-CoV2 infection.
[0060] In some embodiments the sensing test strip that detects the proteases may employ colorimetric reagents that change color only in the presence of the target protease or proteases. For example, in one embodiment the colorimetric reagent may be based on gold nanoparticles (AuNPs). Solutions of gold nanoparticles are normally red. However, when the particles become very close to each other, the solution becomes blue/purple. A suitable peptide can be used to cause electrostatic interactions that ensure that this color change occurs only in the presence of the Mpro protease. For example, as will be described in more detail below, the peptide that is selected may be a modular zwitterionic peptide having the general formula (Asp)n“(AA)x-(Arg)m, with n>m. These peptides have been found to induce AuNP aggregation in response to proteases. In particular, the zwitterionic peptide has both positive and negative building blocks. After Mpro cleavage of the peptide, the positive-charged segments attract negatively-charged gold nanoparticles, thereby yielding a color change.
Examples of suitable peptides include the ZY7, and YR9 peptides, corresponding to varying Arginine (i.e., positive amino acid, shorted in R in single letter) with m = 2, 4 with “n” and “x” fixed.
[0061] While one particular detection mechanism for implementing a colorimetric reagent that changes color only in the presence of the Mpro protease has been discussed above, those of ordinary skill in the art will recognize that other detection mechanisms may be employed instead to detect this particular protease or other proteases (e.g., PLP) that may be chosen as the target biomarker. For example, another detection mechanism employing a different peptide may be found, for example in Jin, Z.; Jorns, A.; Yim, W.; Wing, R.; Mantri, Y.; Zhou, J.; Zhou, J.; Wu, Z.; Moore, C.; Penny, W. F.; Jokerst, J. V. Mapping Aerosolized Saliva on Face
Coverings for Biosensing Applications. Anal. Chem. 2021, 93, 11025-11032, which is incorporated by reference herein in its entirety.
[0062] FIG. 2 shows an exploded view of one particular embodiment of a sensing test strip that may be secured to a face mask in a removable manner. In this embodiment the test strip employs a multilayer structure that includes a cover layer 110 having one or more vents 102 to allow transfer of breath, saliva and aerosols to an underlying absorbent pad 112 that serves to concentrate the proteases. A spacer layer 114 has precut channels in which the absorbent pad 112 is placed. A reagent housing 115 such as a blister pack, which stores the reagent in the liquid phase, is also placed in a precut opening of the spacer layer 114 so that upon release of the reagent (e.g., upon opening of the blister pack by compressing it) it flows into the absorbent pad 112 to interact with the captured biomarkers. The spacer 114, along with the pad 112 and reagent housing 115 is placed onto a support layer 116 that has vents 117 that are aligned with the vents 101 in the cover layer 110. Adhesive on the lower surface of the cover layer 110 and the upper surface of the support layer 116 can be used to secure the spacer 115 to both the cover layer 110 and the support layer 116. The bottom surface of the support layer 116 may also be coated with an adhesive that is covered with a release liner that the user removes when the sensing test strip is to be secured to the face mask.
[0063] The absorbent pad 112 may include two flow channels or paths 112A and 112B, one of which serves as a positive control that is treated with recombinant protease biomarkers. The other flow channel or path will remain untreated and exposed to air. The cover layer 110 is transparent to allow for visual analysis of both flow channels after the reagent has been released from the reagent housing 115. By way of illustration, in one implementation the sensing test strip may be about 4cm in length and 2 cm wide and the reagent housing 115 may be a blister pack that contains about 500 pL of the reagent. More generally, the size, shape and overall structure and configuration of the sensing test strip may be application specific and is not limited to the example described herein.
Proof-of-Concept
[0064] As previously mentioned, the use of the illustrative sensing test strip described above was validated for various face mask types using a-amylase as the biomarker. First, the levels of saliva amylase accumulated on the sensing test strips when affixed
on each face covering using an amylase reagent was measured. These results are shown in connection with Figs. 3A-3F.
[0065] Fig. 3 A shows the overall structure of the sensing test strip that was used, which is largely the same as the test strip described above in connection with Fig. 2. In these experiments the vents on the front cover Pl and the bottom adhesive layer P6 had an area of about 0.72 cm2. Fig. 3B is a white-light image of a test strip affixed to the outside of a neck gaiter and another offset parallel inside the mask. The mask material was cropped as a control sample. Participants (n=10) wore each face covering with the test strips for 8 hours. The measured amylase level on the outside/inside test strips attached to different face coverings are shown for a neck gaiter (Fig. 3C), for a cloth mask (fig. 3D), a surgical mask (Fig. 3E), and a N95 respirator (Fig. 3F). The Y-axis in Figs. 3C-3F denotes the level of salivary amylase, which is proportional to the amount of saliva droplets accumulated on the masks. In addition, the amylase level on the mask materials was also included as a control (the most right in each box plot in Figs. 3C-3F).
[0066] Surprisingly, despite a 95% reduction in the testing area compared to the naked facemasks (0.72 vs. 20 cm2), the test strip placed inside the face coverings still captured enough amylase for detection. For example, the determined amylase levels were 1,166 U/L for the strip placed inside neck gaiter, 533 U/L for that of the cloth mask, and 2,200 U/L for the strip inside surgical mask. When vertically affixed to the N95 chamber, however, the test strip had low efficiency of saliva collection (300 U/L). This could arise from the large breathing room dispersing the amylase at a low amount per area. Additionally, the presence of amylase on the inner strips has a good concordance with that of mask controls (/?>0.05). No amylase was detected on the strips placed outside the face coverings. Accordingly, these results suggest that it generally may be preferable to place the test strip on the inner surface of the face mask, although there may nevertheless be situations, in combination with certain mask types, in which it may nevertheless by possible and advantageous to place it on the outer surface, or between layers of a mask having a multilayer body.
[0067] Next, as shown in Figs. 4A and 4B, a proof-of-concept example of a mask was provided with the capability of detecting amylase in situ. Fig. 4A shows that the sensing test strip that was used was based on the design shown in Fig. 3 A, except that an extra layer of forensic press paper P4 was inserted underneath the polyester pad P5. The press paper P4 acts as a blue color agent. The inset in FIG. 4A shows the detection
mechanism. The blister pack P5 that serves as the reagent storage was loaded with deionized water. After release of the water, the transmitted amylases release the blue dye from the microsphere surfaces to both the absorbent pad and the forensic paper in the presence of the water media, yielding a color change on both the front and back side of the sticker. Experimentally, the test strip was attached inside a neck gaiter for 8 hours. Fig. 4B shows white-light images of the amylase-sensing test strip before and after release of the water. The test line on the pad shifted its color from white to blue (represented by the darker regions in Fig. 4B), which can be viewed from both the front and back sides. For reference, the sealed control line without exposing to any oral droplets retained its original color in white.
[0068] Additional details concerning the experiments that were conducted to validate the techniques described herein may be found in Jin, Z.; Joms, A.; Yim, W.; Wing, R.; Mantri, Y.; Zhou, J.; Zhou, J.; Wu, Z.; Moore, C.; Penny, W. F.; Jokerst, J. V. Mapping Aerosolized Saliva on Face Coverings for Biosensing Applications. Anal. Chem. 2021, 93, 11025-11032, which is incorporated by reference herein in its entirety.
[0069] Additional details and experimental results are provided below concerning the use of a zwitterionic peptide that carries opposite charges at the C-/N-terminus to exploit the specific recognition by Mpro. Among other things, it is shown that a limit of detection for Mpro in breath condensate matrices is less than 10 nM. In addition, it is shown that among ten COVID-negative subjects who were assayed, no results were false-positive. These and further details also may also be found in Jin, Z.; Mantri, Y.; Retout, M.; Cheng, Y.; Zhou, J.; Joms, A.; Fajtova, P.; Yim, W.; Moore, C.; Jokerst, J., A Charge-Switchable Zwitterionic Peptide for Rapid Detection of SARS-CoV-2 Main Protease. Angew. Chem., Int. Ed., 2021. DOI: 10.1002/anie.202112995, which is incorporated by reference herein in its entirety.
Charge- Switchable Zwitterionic Peptides
[0070] Colorimetric sensors based on gold nanoparticles (AuNPs) are popular because of their unique photophysical properties including a surface plasmon resonance (SPR) band with a high molar absorption coefficient. Aggregation enhances the near-field interactions of proximal nanoparticles and leads to a bathochromic shift in the SPR band; this in turn leads to a pronounced color change. Strategies for plasmonic coupling can be grouped into chemical linking and physical factors. For example, dithiothreitol is a simple molecule to cluster AuNPs due to the
strong thiol-to-gold bond. Nanoplasmonic sensing based on this covalent bond have been reported for thrombin, trypsin, furin, etc.
[0071] Alternatively, controlled physical factors (e.g., ionic strength, solvent polarity, and ligand hydrophilicity) can also impact colloidal dispersion. In particular, nanoparticles primarily stabilized through electrostatic double layer repulsions tend to aggregate via the addition of excess salt due to charge screening. Tuning non-specific electrostatic interactions has also led to several simple and rapid colorimetric assays for heavy metal ions. In principle, these electrostatic processes could be repurposed for sensing other targets (here, proteases) by rationally designing the electrophoretic property of a specific substrate where the selectivity is based on molecular recognition between the enzyme and the substrate.
[0072] Here we report on a general formula of modular zwitterionic peptides, (Asp)«-(AA)x-(Arg)m, that induce AuNP aggregation in response to proteases. We then apply this system to colorimetrically measure Mpro. Unlike nucleic acids testing, protease detection uses autolytic cleavage of the substrates that amplifies signals without the polymerase chain reaction. The key role of Mpro in the propagation of infectious SARS-CoV-2 — in addition to a lack of human homologues — make this protease an ideal maker for COVID-19 diagnosis.
[0073] In our system, the modular zwitterionic peptide has oppositely charged termini, /.< ., (Asp),, and (Arg)m, flanking a specific Mpro cleavage site (AA)X. Because n > m, the intact peptide carries a net neutral charge. Accordingly, the interactions between the intact peptide and sulfonated ligand-coated AuNPs are minimized given the negatively charged sulfonate groups. Proteolytic segmentation of the peptide would switch the electrophoretic property of the corresponding fragment, thus triggering the collapse of gold suspension via electrostatic attractions. This induces a visible color change. By using a combination of the zwitterionic peptide and AuNPs in label-free conditions, we showed that this approach can rapidly detect Mpro at approximately 3 to 10 nM in buffer and exhaled breath condensate in 10 min. Several mutated peptides were built to investigate the sensing mechanism and to optimize the sensor performance. Finally, we reframed the sensor configuration by building it into a lateral flow strip to create portable sensing devices as a point-of-care surveillance tool for CO VID disease. Overall, the novel peptide-enabled colorimetric assay does not require interfacial bioconjugations or any specific instrumentations, and thus may have value in surveillance of CO VID.
[0074] Fig. 5A schematically shows the intact peptide and its proteolytic fragments with distinct physicochemical properties (e.g., net charge, interfacial affinity) to modulate the dispersity of a gold suspension. In particular, Fig. 5A shows a schematic illustration of the aggregation of bis(/?-sulfonatophenyl)phenylphosphine-coated gold nanoparticles (BSPP-AuNPs) caused by the proteolytic hydrolysis of the intact peptide, where the net charge of intact peptide and its fragment is reversed. The cartoon designates proteases, /.< ., Mpro; the tandem hexagon represents a modular zwitterionic peptide with a Mpro recognition site. We hypothesize that successful proteolysis of the substrate will induce clustering of the monodispersed particles, which changes the color from ruby red to violet-blue by naked eye. In addition, as illustrated in Fig. 5B, this color change reporter when integrated with routinely-used mask could allow one to easily monitor the COVID-infectious status.
[0075] Fig. 5B shows that the colorimetric test could be coupled with face coverings with a lateral flow strip to indicate CO VID infection in-situ (top). White-light image of BSPP-AuNPs after incubating with the proteolytic products. The visual color shifts from ruby red to violet-blue (indicated in gray-scale in Fig. 5B) with increasing amount of Mpro from 0-200 nM (bottom). Fig. 5C show TEM images of the dispersed BSPP-AuNPs (top) and proteolysis-induced gold aggregates [bottom, (i)]. The flocculated AuNPs can be redispersed using ionic surfactant additives, such as sodium dodecyl sulfate [SDS, (ii)], due to the restored electrostatic repulsion.
[0076] The zwitterionic peptides were rationally designed to encompass arranging three functional domains in a linear N-to-C-terminal configuration (Fig. 6A), including: (i) an N-terminal charge shielding domain made of repeating aspartic acid (Asp, D) for neutralizing the charge of the intact peptide, /.< ., part iii; (ii) an exposed protease-recognition sequence; and (iii) a C-terminal aggregating site for clustering gold colloids. The aggregating domain (part iii) has a repeating arginine (Arg, R) and a cysteine (Cys, C) bridge with high affinity to AuNPs via electrostatic and coordinating interactions. Overall, the peptide design meets two key requirements. The first is little or no attraction between the intact peptide and AuNPs. This is achieved by matching the charge state of intact peptide to that of particle surfaces, which typically originates from the terminal groups of the surface ligand. Second, peptide fragmentation by the protease exposes the aggregating domain that favors tight self-assembly of particles. This modular design is also intended to obviate the need for major revisions when targeting a new protease. That is, the specificity of the
sensor is tuned by changing only the cleavage site while keep the other components the same. In addition, the inclusion of aspartic acid and arginine residue improves the hydrophilicity of the oligopeptide and ensures the sensor performance in aqueous media. Fig. 6A shows a representative peptide ZY7 synthesized based on the above criteria: DDDTSAVLQSGFRCGRGC (net charge = 0). Specifically, ZY7 contains a Mpro cleavage sequence LQJ.SG [i.e., at C-terminus of glutamine (Q)] surrounded by a negatively and positively charged moiety (i.e., DDD vs. RCGR). To the best of our knowledge, no mammalian protease cleaves after a C-terminal glutamine. Hence this cleavage site is unique for viral proteases only. Proteolysis of ZY7 peptide by Mpro has been confirmed by HPLC and ESI-MS and results in the formation of two fragments: DDDTSAVLQ (net charge = -3) and SGFRCGRGC (net charge = +3, with a dithiol bridge); see Fig. 6B and Table 1.
[0077] Fig. 6B shows HPLC and ESI-MS data showing that Mpro selectively cleaves the ZY7 peptide at the C-terminus of glutamine (Q). The peak with * is the product. See also Table 1 for additional information concerning the ZY7 and other peptides. [0078] The functional peptide is used under label-free conditions with AuNPs as a readout to detect the presence of Mpro. This is advantageous because surface labelling techniques often require precise control over the bio-nano functionalization and may involve frequently unanticipated particle aggregation. Here, we chose bis( - sulfonatophenyl)phenylphosphine (BSPP) to modify the 13-nm citrate-AuNPs made by Turkevich method, which fulfill with two guiding parameters. First, the BSPP ligand is rich in terminal sulfonate groups and thus gives the nanoparticle strong negative charges, i.e., the zeta potentials (Q are -26.5 ± 1.5 mV. Second, the nanoparticles are colloidally stable in various media due to the effective phosphorus- to-gold coordination, large steric hindrance.
Table 1. Peptide information and limit of detection (LoD) for Mpro.
Arg and
Cys DDDTSAVLQ SGFRCGR 27.
ZY7 1884.8 0 3.2 -55.3 33.4 68.4 bridge,
GC ; - 7 zwitterio n scramble DDDTSAVLExSGFRCGR
MS7 1855.8 1 sequenc
GC e
DDDTSAVLQJ.SGFACG Cys
NR10 1714.6 -2 >100 __ __
AGC bridge no
DDDTSAVLQ SGFAGG
NC11 1622.7 -2 >100 __ __ __ Arg/Cys
AGG bridge
D-amino
DDDTSAVLQ^SGFrcGrG 114 acid
DS12 1884.3 0 2.7-73.2 94.2 142.4 .4 substitut ion no
TE3 RGRTSAVLQJ.SGFRC 1535.8 +4 2.5 -3.1 -- -- -- shieldin g site single
OR8 DDDTSAVLQJ.SGFR 1408.6 -1 >100 __ __
Arg
DDDGDDDTSAVLQJ.SG quadrupl
YR9 2336.1 -1 0.3 -5.1 3.4 9.5
FRRGRR e Arg
YF15 SGFRRGRR 989.6 +5 <0.3 __ __ __ fragment
[a] The electrophoretic property at pH 8.0; note that all the peptides contain a free N-terminus and an amidated C-terminus. [b] The operation window was determined in Tris-buffer (TB) media at 10 min readout time, [c] EBC: exhaled breath condensate, [d] J, designates the Mpro cleavage site; x designates unrecognized site for Mpro. [e] The single-letter in uppercase is L-amino acid (AA), while the singleletter in lowercase is D-amino acid.
from the bulky aromatic rings, and strong electrostatic double layer repulsions. BSPP- capped AuNPs have a compact ligand layer and thus favor tight interparticle interactions, e.g., the hydrodynamic diameter (DH) is 19.1 nm with a PDI of 0.10. [0079] We here selected Mpro peptidase as a model example to test our label-free peptide and BSPP -AuNPs. Mpro regulates viral replication and transcription and is therefore an attractive therapeutic and diagnostic target for SARS-CoV-2. We first validated our hypothesis by incubating BSPP-AuNPs with either ZY7 parent peptide or its pre-digested fragments. The full cleavage was confirmed by HPLC and ESI-MS (Fig. 6B). Fig. 6C shows the color change (in gray scale) as a function of the peptide concentration and time. The addition of ZY7 fragments (6-50 pM) to the BSPP gold dispersion caused an instant color shift from ruby red to purple. Such a color evolution continuously built with increasing fragments concentration and reaction time. In comparison, BSPP-AuNPs coexist with the ZY7 parent peptide with no change of the color.
[0080] Characterizations of the gold aggregates and several peptide derivatives of ZY7 were subsequently employed for a mechanistic study. TEM images in Fig. 5C and hydrodynamic diameter (DH) by dynamic light scattering (DLS) clearly indicate the formation of AuNP aggregates in the presence of ZY7 fragments. In particular, Fig. 6D shows DLS profiles of BSPP-AuNPs (3.8 nM, 100 pL) incubated with increasing concentrations of ZY7 parent peptide (darker circles) and ZY7 fragments (lighter circles). No change of the hydrodynamic diameter (DH) for BSPP-AuNPs was observed when incubated with ZY7 peptide, while such a change became sizable in the presence of ZY7 fragments of more than ~3 pM. The size increases commensurate with alternations in surface charge. For example, the zeta potentials of BSPP-AuNPs mixed with increasing amount of ZY7 fragments showed a sizable increase from original value to nearly neutral, e.g., -26.5 vs. -2.5 mV; see Fig. 6E, which shows zeta potential measurements of AuNPs (3.8 nM, 100 pL) incubated with increasing concentrations of ZY7 parent peptide (darker circles) and ZY7 fragments (lighter circles). The ZY7 fragments adsorbed to the particle and altered the surface charges from -26.5 to -2.5 mV. Error bars represent triplicate measurements for one sample. . We attribute this increasing charge to the positive guanidine groups in arginine-rich fragment adsorbed onto the surface sulfonate via electrostatic attractions. This
neutralized the surface charge and compromised the interparticle repulsions thus resulting in the collapse of colloidal system.
[0081] To confirm this, we synthesized an analogue peptide NR10, where the arginine residues were substituted with neutral alanine; see Table 1. In this case, the absence of arginine determinants caused no aggregation of the AuNPs irrespective of proteolytic cleavage. This manifested in no change in DH (i.e., approx. 20 nm) and zeta potentials, albeit there was a slight decrease in the surface potential due to weak association of NR10 of -2 charge on the particle surfaces (i.e., -30 vs. -40 mV). We therefore conclude that the aggregation of BSPP- AuNPs was dominated by the positively charged arginine residues rather than disulfide bridging. To this end, we quantitatively assessed the color changes through analyses of the progression of UV- Vis spectra. Figs. 6F-6G show the time progression of optical absorption of AuNPs (3.8 nM, 50 pL) when incubated with ZY7 parent peptide and its fragments (c = 6.0 pM). The curves were recorded every 10 min for 2 h. Noticeable peak changes were observed at 520 and 600 nm for a fragment- AuNP mixture. In Figs. 6F-6G, the time- lapsed absorbance profiles of AuNPs with ZY7 parent peptide remain essentially the same indicating a preserved dispersity. The ZY7 fragment led to a sizable decrease in the SPR at 520 nm commensurate with a noticeably raising band at 600 nm. This is a clear indication of AuNP aggregation and color change. We thus define a ratiometric signal, Abs6oo/Abs52o, to quantify the aggregation and color change.
[0082] The above mechanism favors electrostatic-induced flocculation of AuNPs and was further corroborated by a reversible colorimetric experiment, employing several different surfactants. For example, the addition of ionic surfactants such as cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) recovered colloidal AuNPs from the aggregates. Excess anionic SDS scavenges the positively charged peptide fragments, thus restoring the electrostatic double layer repulsions between particles.
[0083] To study the limit of detection (LoD) for Mpro based on our sensing system, we first optimized the concentration of ZY7 peptide in an assay. Experimentally, BSPP -AuNPs (3.8 nM, 100 pL) were incubated with peptides and the optical measurements (Abseoo/Abs52o) were recorded at 10 min after AuNP addition. The minimum amount of fragment needed to induce a noticeable color change has to exceed 3.2 pM, but parent ZY7 should not cause particle aggregation and only
slightly affects the particle dispersity at concentrations higher than 55.3 pM (see Fig. 7A, which shows the ratiometric signal (Abseoo/Abs52o) collected from BSPP-AuNPs (3.8 nM, 100 pL) incubated with various amount of ZY7 parent and fragments. The working window for ZY7 substrate is 3.2-55.3 pM.). Excess ZY7 peptide likely increases the ionic strength and screens the repulsive forces between particles thus making the system colloidally unstable. Accordingly, we defined a working window for the assays based on ZY7 as 3.2-55.3 pM; see Table 1. In practice, a concentration of 50 pM has been observed to yield the best result and was defined as the optimal peptide concentration for the rest of this study.
[0084] To understand the clinical value of the system, we tested Mpro detection in three different matrices including Tris buffer (TB), exhaled breath condensate (EBC), and diluted pooled saliva (50%). The enzyme assay was performed by incubating a defined concentration of ZY7 substrate (i.e., 50 pM) with various concentrations of spiked Mpro (i.e., 0-200 nM) in a 20 pL volume for 3 h. Subsequently, 100 pL of BSPP gold dispersion (3.2 nM) was added as a readout mechanism. All concentrations are with respect to a 120 pL volume; see more details in Supporting Information. Representative aggregation kinetics in EBC within 1 hour are shown in Fig. 7B. In particular, Fig. 7B shows the time progression of absorbance ratio in the enzyme assay, where a fixed amount of ZY7 substrate (50 pM) is incubated with increasing concentrations Mpro (0-200 nM). The test media is exhaled breath condensate (EBC). The data points were read every 1 min for 1 hour. The trend clearly indicates that higher amount of Mpro led to more particle aggregations or rapid color change and vice versa. Importantly, BSPP-AuNPs were stable in the presence of Mpro alone and in all tested buffers.
[0085] Next, we set 10 min as the readout time for pursuing a rapid protease detection. Fig. 7C shows the ratio of Abseoo/Abs52o as a function of Mpro concentration. Thus, the LoD for Mpro was determined to be: 27.7 nM in Tris buffer (TB), 33.4 nM in EBC matrix, and 68.4 nM in 50% saliva. The method for LoD calculation is reported in previous literature. This suggests that our sensor is best performing in TB and EBC media, while other complex matrices such as saliva would yield sensitivity in about 3-fold lower or more. No aggregation occurred in human plasma presumably negative proteins that scavenge the positively charged peptide fragments. The clinical-relevant level of Mpro in novel SARS-CoV-2 infected cells
still lacks rigor reports. Nevertheless, the protease LoD of our sensor is similar to other nanoparticle-based protease assays (i.e., in low nanomolar ranges), but is much less time-consuming (/.< ., t =10 min). On the contrary, control experiment using a scrambled peptide sequence, MS7, showed no AuNP aggregation at any concentrations of Mpro (see Fig. 7D, which shows the absorbance ratio as a function of Mpro concentration in three biofluids, where MS7 (control) substrate (50 pM) is used. Readout time is set to be 10 min.). For MS7 peptide, we substituted the indispensable glutamine (Q) at Pl site with its analogue, glutamic acid (E), and therefore no specific recognition by Mpro, circumventing the formation of aggregating fragments; see Table 1. Importantly, the colorimetric detection can be carried out in a one-pot assay. The enzymatic cleavage and particle aggregation took place simultaneously in-situ, albeit a longer readout time was needed (~1 h). The LoD of the one-pot assay for Mpro is about 40.9 nM.
[0086] We briefly describe the perspective to improve our protease detection limit. The above modulation of particle dispersity is based on the electrophoretic property of substrates, and thus we further explored the effect of tuning arginine number from 0, 1, 2, and 4, which corresponds to NR10, OR8, ZY7, and YR9 peptide, respectively (Table 1).
[0087] FIGs. 8 A, 8B and 8C show the operation window of Mpro sensors based on NR10, OR8, and YR9 peptide, which contains 0, 1, and 4 arginine residues in its aggregating sequence, respectively. According to Figs. 8A and 8B, the aggregating sequences containing zero or single guanidine side chain produce no optical signal change. These non-aggregating systems have low ionic valence and attenuated electrostatic interactions according to the Schulze-Hardy rule. Compared to the ZY7 fragment of two arginine residues, the inclusion of four arginine residues in YR9 strongly interferes with the particle surface potential and requires substantially lower concentrations to induce aggregations (e.g., 0.3 vs. 3.2 pM, Fig. 8C). This also resulted in an eight-fold improvement in sensitivity to Mpro (see Fig. 8D, which shows the Mpro LoD of sensors based on four peptides of varying number of arginine: no LoD observed for NR10 and OR8 substrate; 27.7 and 3.4 nM is determined for YR9 and ZY7 substrate, respectively.). That is, the LoD is improved from 27.7 to 3.4 nM in TB by using YR9 peptide of high ionic valence (Table 1).
[0088] Next, we used a known competitive inhibitor (GC376) for Mpr0to study the inhibition assays. Fig. 8E shows the results of assaying an increasing molarity of GC376 (i.e., 0-1 pM) in the presence of constant amount of Mpro (100 nM) and ZY7 substrate (50 pM). Note that the inhibitor itself does not affect the particle dispersity; see the control line in Fig. 8E. As anticipated, the aggregation kinetics were largely retarded due to low activities of pre-mixed Mpro with the inhibitor. Examination of the absorbance ratio at 15 min yields a typical inhibitor titration curve (see Fig. 8F, which shows a typical inhibition titration curve fitted with the Morrison equation for the competitive inhibitor, GC376, where the inset shows the chemical structure of GC376 inhibitor). A linear form of the Morrison equation derived by Henderson was applied to evaluate the titrated Mpro concentration ([E]o=6O nM) and the potency of GC376 inhibitor [Ki (apP) =15 nM, IC50 =45 nM], This half maximal inhibitory concentration (IC50) is lower than the majority of reported values most likely due to the different assay conditions, such as incubation time and buffer pH. Overall, this inhibitor assay demonstrated that our sensing system can be employed to rapidly screen for anti-Mp'° therapeutic agents.
[0089] We further cross-tested several mammalian proteins against our sensing system, including bovine serum albumin (BSA), hemoglobin, a-amylase (z.e., digests a-l,4-glucosidic bond in starch), thrombin (z.e., cleaves Arg-Gly bond in fibrinogen), and trypsin (z.e., cleaves at C-terminus of Arg or Lys). Fig. 8G shows sensor activation by other mammalian proteins (100 nM), including bovine serum albumin (BSA), hemoglobin, a-amylase (100 U/mL), thrombin, and trypsin. Assay with and without Mpro is included as positive and negative control. As shown in Fig. 8G, no particle aggregation was detected in the presence of BSA and hemoglobin, indicating good compatibility of the sensor with common proteins. Similarly, no optical signal was addressed by addition of other enzymes such as a-amylase or thrombin. Nonetheless, arginine-specific proteases such as trypsin caused false positive results. [0090] ESI-MS data confirmed that ZY7 substrate was recognized by trypsin at the C-terminal of arginine at P4’ site, exposing a short CGRGC fragment that causes the AuNP aggregation. Indeed, this short fragment was also found when the assay was performed in saliva leading to background signal (the pooled saliva could contain Arg-gingipains implicated in periodontal disease). As a viable strategy to suppress such a side effect and use our sensor in other clinical samples containing trypsin-like
enzymes, we mutated the aggregating amino acids with their D-enantiomers to create a new DS 12 peptide; see Table 1. The results assaying from the DS 12 substrate showed improved proteolytic resistance to trypsin (see Fig. 8H, which shows the response of sensors based on ZY7 and DS12 substrate to Mpro or trypsin in Tris buffer. LoD for MPro is 27.7 and 114.4 nM for ZY7 and DS12, respectively; LoD for trypsin is 9.7 and 30.3 nM for ZY7 and DS 12, respectively.). Remarkably, no background activation was observed for the sensor based on DS 12 in saliva media. Despite these expectations, the LoD for Mpro using DS 12 substrate showed an approximately 3-fold reduction comparing to that of ZY7 substrate (Table 1). The loss in sensitivity is in alignment with previous reports of a 4-fold reduction in Mpro efficiency on alteration of the P4’ arginine.
[0091] The relevance of peptide/ AuNP -based protease detection resides in its simplicity where no complex bioconjugation techniques or readout instrumentation are required. This allows for the large-scale implementation of qualitive diagnostics for protease markers. As previously discussed, tests like these can be easily affixed to routinely used face coverings, turning them into easy-to-use diagnostic kits. As also described earlier, the detection technique described herein can be adapted onto a flowcell inspired Mpro sensing strip of the type described above in connection with Fig. 2 and further shown in Fig. 9A and Fig. 9B, which shows a white light image of the assembled sensing test strip, equipped with a BSPP-AuNP dispersion in the blister pack, ZY7 peptide on the test lane (left, dash box), and YF15 peptide on the positivecontrol lane. The test strip consisted of two pad lanes: (i) A test lane decorated with 50 pM of ZY7 peptide, and (ii) a positive control lane loaded with 50 pM of peptide fragment (YF15). This peptide fragment is the most potent aggregating domain from all the peptides listed in Table 1. 60 pL of BSPP-AuNPs was housed in the blister pack and used for readout or color agent (Fig. 9B). In principle, one pink and one purple lane would signify a COVID-negative test, while two purple lanes indicate a positive test for the presence of Mpro.
[0092] We determined the LoD for Mpro on the sensing strip. As illustrated in Fig. 9C, which shows various concentrations of Mpro in EBC (0-100 nM, with respect to 30 pL volume) tested for 10 min. The test lanes incubated with Mpro more than 40 nM started to appear as purple color, indicating positive test results. We thus conclude that the sensor LoD for Mpro is similar in both solution and semi-solid (i.e., polyester
pad) phase, 30-40 nM. Figs. 9E and 9F show SEM images of non-aggregating AuNPs on the red lane and clustered AuNPs on the purple lane, respectively.
[0093] In a real scenario for a COVID-infected patient, the strip/pad affixed on a face covering would collect the respiratory droplets that harbor Mpro biomarker (for 8 h or a normal workday). Meanwhile, on the absorbent pad the concentrated Mpro proteases start to cleave the pre-loaded ZY7 peptide. At the end of the day, the BSPP-AuNPs in the blister pack are released and moved to the absorbent pad via capillary action to interact with the peptides for 10 min readout. As a proof-of-concept experiment, we show here ten EBC samples collected from the COVID-negative volunteers (/.< ., verified by PCR test) and showed no false positives (see Fig. 9D, which shows strip testing of the Mpro marker on COVID-negative participants (NP#, 77=10). All test lanes show pink-red in the absence of the SARS-CoV-2 protease. FIG. 9E shows an SEM image of non-aggregating AuNPs on the red lane and FIG. 9F shows an SEM image of clusters AuNPs on the purple lane. This result was also collaborated with solution-phase assays where no alterations in ratiometric signals were detected (see Fig. 9G, which shows sensor testing on aqueous EBC matrices collected from COVID-negative subjects (w=10). No absorbance ratio change/false positive was noticed).
[0094] In summary, a universal formula of zwitterionic peptide, (Asp)«-(AA)x-(Arg)m, is reported to modulate the dispersity of AuNPs and applied for a colorimetrical detection of Mpro, a protease implicated in SARS-CoV-2 viral replication. We built a label-free peptide (ZY7) that carries net neutral charge causing no aggregation of negatively charged BSPP-AuNPs. While enzymatic cleave of ZY7 by Mpro releases a positively charged fragment that triggers the collapse of gold suspension via electrostatic interactions. By quantifying the color change with measurable absorbance ratio, we have determined that our sensor performs best in EBC with an LoD of 33.4 nM using the ZY7 peptide. This rapid and reproducible color change provides a simple platform for inhibitor-screening targeting at MP10. We assayed ten COVID-negative human subjects and found no false-positive result. Overall, the present AuNP -based colorimetric assay does not require tedious bioconjugations or specific instrumentations, and hence can be easily integrated into qualitative diagnostic kits for daily use.
[0095] The above examples and disclosure are intended to be illustrative and not exhaustive. These examples and description will suggest many variations and alternatives to one of ordinary skill in this art. All these alternatives and variations are intended to be included within the scope of the attached claims. Those familiar with the art may recognize other equivalents to the specific embodiments described herein which equivalents are also intended to be encompassed by the claims attached hereto.
Claims
1. A colorimetric assay, using a stimuli-responsive peptide for detecting an enzyme, comprising: a modular peptide having physicochemical properties that change in response to a presence of an enzyme of interest; and a color reagent indicating a presence and quantitative level of the enzyme of interest when treated with the modular peptide via a visual readout, the quantitative level of modular peptide activation being related to a quantity of the enzyme of interest such that the colorimetric assay is applicable for purposes of inhibitor screening and diagnostic testing.
2. The assay of claim 1, wherein one of the physicochemical properties is charge potential, the modular peptide being a charge-switchable zwitterionic peptide having a general formula (Asp/Glu)«-(AA)x-(Arg/Lys)m, and being synthesized via a Fmoc- SPPS method, wherein an (Arg/Lys)m block of the modular peptide includes at least Arg and Lys units and an (Asp/Glu),, block of the modular peptide includes at least Asp and/or Glu units.
3. The assay of claim 2, wherein the color reagent includes negatively-charged-ligand functionalized plasmonic nanoparticles.
4. The assay of claim 3, wherein, when intact, the peptide is in a net neutral or negative charge state that has minimal interactions with the functionalized plasmonic nanoparticles, with n > m > 2.
5. The assay of claim 2, wherein -(AA)x- is an amino acid sequence of varying length comprising natural and synthetic amino acids that is responsive to the enzyme of interest, the enzyme of interest being selected from the group consisting of a viral protease, a mammalian protease, or a combinations thereof.
6. The assay of claim 3, wherein installed ligands are phenyl phosphine derivatives selected from the group consisting of: diphenylphosphinobenzene sulfonate (DPPS),
-27-
bi s(p-sulfonatophenyl)phenylphosphine (B SPP), triphenylphosphine-3 ,3 , 3 - trisulfonate (TPPTS), and combinations of the above.
7. The assay of claim 3, wherein a charged ligand is installed on plasmonic nanoparticles by a ligand exchange or phase transfer reaction.
8. The assay of claim 3, wherein plasmonic particles of the negatively-charged-ligand functionalized plasmonic nanoparticles are prepared from citrate-stabilized spherical gold nanoparticles by a Turkevich method.
9. The assay of claim 4, wherein a negative charge on the peptide arises from aspartic acid, glutamic acid, or a combination of thereof and a positive charge arises from arginine, lysine, or a combination thereof.
10. The assay of claim 4, wherein the charge-switchable zwitterionic peptide is used with gold nanoparticles under label free conditions.
11. The assay of claim 6, wherein a mass concentration for ligand exchange is 0.5 to 2 mg of ligand per mL of nanoparticle volume.
12. The assay of claim 2, wherein the enzyme of interest is a protease, a peptide fragment arising from protease breakdown being a positively-charged species that electrostatically interacts with gold nanoparticles.
13. The assay of claim 12, wherein an electrostatic attraction of the gold nanoparticle induces a plasmonic coupling and color changes.
14. The assay of claim 12, wherein a color change arising from nanoparticle aggregation occurs either after introducing the protease pre-mixed with the modular peptide to a dispersion of the gold nanoparticle or after introducing the protease to the modular peptide premixed with the gold nanoparticles.
15. The assay of claim 14, wherein incubation of the protease with the modular peptide occurs at 37 °C for 0.5 to 3 hours, with a molar ratio of [peptide]: [protease] varying from 250 to 100,000.
16. The assay of claim 14, wherein color changes at 10 min are recorded by eye or using a smart phone, an optical absorption spectrometer, or both.
17. The assay of claim 14, wherein the gold nanoparticles are stable in buffers for no less than 24 hours.
18. The assay of claim 15, wherein a sample to be analyzed contains biological samples.
19. The assay of claim 18 wherein the biological samples are selected from the group including exhaled breath condensate, saliva, human plasma, urine and gingival crevicular fluid.
20. The assay of claim 19 wherein the biological samples further include buffers.
21. The assay of claim 20 herein the buffers include dithiothreitol.
22. The assay of claim 15, wherein a detection limit of the protease is at a first molar ratio of [peptide]: [protease] that shows an observable color change.
23. The assay of claim 12, wherein an amount of gold nanoparticles used for reporting a color change is 100 pL of 3.4 nM.
24. The assay of claim 22, wherein the detection limit is tunable by changing a value of m such that increasing the value of m improves the detection limit of the protease.
25. The assay of claim 1, wherein one of the physicochemical properties is interfacial affinity, the modular peptide being an affinity-switchable divalent peptide having a general formula (Asp)«-(AA)x-(Cys)m, and being synthesized via a Fmoc-SPPS
method, an (Asp),, block of the modular peptide including at least Asp units and a (Cys)m block of the modular peptide including at least Cys units.
26. The assay of claim 25, wherein n >2, and m = 2.
27. The assay of claim 19, wherein the modular peptide combines with gold nanoparticle modified with citrate or tris(2-carboxyethyl)phosphine (TCEP) ligand.
28. The assay of claim 1, wherein one the physicochemical properties is amphiphilicity, the modular peptide being an amphiphilicity-switchable peptide having the general formula (Asp)«-(A A)x-(Phe-Phe-Pro)m-Cys, synthesized via a Fmoc-SPPS method, an (Asp),, block of the modular peptide including at least Asp units.
29. The assay of claim 28, wherein n >2, and m > 1.
30. The assay of claim 28, wherein the peptide combines with gold nanoparticles modified with citrate or a diphenylphosphinobenzene sulfonate (DPPS) ligand.
31. A method for screening a drug inhibitor, comprising: a. incubating drug molecules of various concentrations with a protease of interest; b. performing colorimetric assays based on a mixture of a peptide and gold nanoparticles in a multiple well plate; and c. measuring an absorbance ratio or color change for an indication of drug potency; and d. wherein the peptide is selected from the group including (i) a charge-switchable zwitterionic peptide having a general formula (Asp/Glu)«-(AA)x-(Arg/Lys)m, and being synthesized via a Fmoc-SPPS method, wherein an (Arg/Lys)m block of the peptide includes at least Arg and Lys units and an (Asp/Glu)„ block of the charge- switchable zwitterionic peptide includes at least Asp and/or Glu units, (ii) an affinity- switchable divalent peptide having a general formula (Asp)«-(AA)x-(Cys)m, and being synthesized via a Fmoc-SPPS method, an (Asp),, block of the affinity-switchable divalent peptide including at least Asp units and a (Cys)m block of the affinity -
switchable divalent peptide including at least Cys units, and (iii) an amphiphilicity- switchable peptide having a general formula (Asp)«-(AA)x-(Phe-Phe-Pro)m-Cys, synthesized via a Fmoc-SPPS method, an (Asp),, block of the amphiphilicity- switchable peptide including at least Asp units.
32. The method of claim 31, wherein the drug molecules are incubated with the protease of interest for 10 to 30 min prior to incubation with the peptide.
33. The method f claim 31, wherein the gold nanoparticles are stable with the drug molecules at testing concentrations.
34. A diagnostic test strip, comprising: an absorbent pad on which a peptide is loaded and fixed; a blister pack having a gold nanoparticle dispersion sealed therein such that upon release from the blister pack the gold nanoparticle dispersion is fluidically communicated onto the absorbent pad, a peptide being selected such that a selected protease concentrated and incubated on the absorbent pad causes a visual indicator to indicate a presence of the selected protease; and at least one carrier layer for supporting the absorbent pad and the blister pack; and wherein the peptide is selected from the group including (i) a charge-switchable zwitterionic peptide having a general formula (Asp/Glu)«-(AA)x-(Arg/Lys)m, and being synthesized via a Fmoc-SPPS method, wherein an (Arg/Lys)m block of the peptide includes at least Arg and Lys units and an (Asp/Glu)„ block of the charge- switchable zwitterionic peptide includes at least Asp and/or Glu units, (ii) an affinity- switchable divalent peptide having a general formula (Asp)«-(AA)x-(Cys)m, and being synthesized via a Fmoc-SPPS method, an (Asp),, block of the affinity-switchable divalent peptide including at least Asp units and a (Cys)m block of the affinity- switchable divalent peptide including at least Cys units, and (iii) an amphiphilicity- switchable peptide having a general formula (Asp)«-(AA)x-(Phe-Phe-Pro)m-Cys, synthesized via a Fmoc-SPPS method, an (Asp),, block of the amphiphilicity- switchable peptide including at least Asp units.
-31-
35. The diagnostic test strip of claim 34 wherein the carrier layer includes an adhesive layer, the diagnostic test strip being configured to be removably securable to a face mask using the adhesive layer such that the selected protease concentrates on the absorbent pad from exhaled breadth condensate or saliva from a wearer of the face mask.
36. A method of making a diagnostic test strip, comprising: loading and fixing a peptide on an absorbent pad; sealing a gold nanoparticle dispersion in a blister pack; arranging the blister pack to cooperate with the absorbent pad so that upon release from the blister pack the gold nanoparticle dispersion is fluidically communicated onto the absorbent pad, a peptide being selected such that a selected protease concentrated and incubated on the absorbent pad causes a visual indicator to indicate a presence of the selected protease; and securing the absorbent pad and the blister pack on at least one carrier layer; and wherein the peptide is selected from the group including (i) a charge-switchable zwitterionic peptide having a general formula (Asp/Glu)«-(AA)x-(Arg/Lys)m, and being synthesized via a Fmoc-SPPS method, wherein an (Arg/Lys)m block of the peptide includes at least Arg and Lys units and an (Asp/Glu)„ block of the charge- switchable zwitterionic peptide includes at least Asp and/or Glu units, (ii) an affinity- switchable divalent peptide having a general formula (Asp)«-(AA)x-(Cys)m, and being synthesized via a Fmoc-SPPS method, an (Asp),, block of the affinity-switchable divalent peptide including at least Asp units and a (Cys)m block of the affinity- switchable divalent peptide including at least Cys units, and (iii) an amphiphilicity- switchable peptide having a general formula (Asp)«-(AA)x-(Phe-Phe-Pro)m-Cys, synthesized via a Fmoc-SPPS method, an (Asp),, block of the amphiphilicity- switchable peptide including at least Asp units.
37. A method of diagnostic testing, comprising: releasing a gold nanoparticle dispersion onto an absorbent pad loaded with a peptide after protease-containing patient samples have been concentrated and incubated on the absorbent pad; and
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after releasing the gold nanoparticle dispersion, determining if a selected protease is present based on a change in visual appearance of the absorbent pad and wherein the peptide is selected from the group including (i) a charge-switchable zwitterionic peptide having a general formula (Asp/Glu)«-(AA)x-(Arg/Lys)m, and being synthesized via a Fmoc-SPPS method, wherein an (Arg/Lys)m block of the peptide includes at least Arg and Lys units and an (Asp/Glu)„ block of the charge- switchable zwitterionic peptide includes at least Asp and/or Glu units, (ii) an affinity- switchable divalent peptide having a general formula (Asp)«-(AA)x-(Cys)m, and being synthesized via a Fmoc-SPPS method, an (Asp),, block of the affinity-switchable divalent peptide including at least Asp units and a (Cys)m block of the affinity- switchable divalent peptide including at least Cys units, and (iii) an amphiphilicity- switchable peptide having a general formula (Asp)«-(AA)x-(Phe-Phe-Pro)m-Cys, synthesized via a Fmoc-SPPS method, an (Asp),, block of the amphiphilicity- switchable peptide including at least Asp units.
38. The method of claim 37 wherein the gold nanoparticle dispersion is stored in a blister pack, the blister pack and the absorbent pad being incorporated in a test strip.
39. The method of claim 38 further comprising applying the test strip onto a face mask such that the protease-containing patient samples are obtained from exhaled breadth condensate or saliva of a wearer of the face mask.
40. The diagnostic test strip of claim 34 wherein the selected protease is Mpro.
41. The method of claim 36 wherein the selected protease is Mpro.
42. The method of claim 37 wherein the selected protease is Mpro.
43. The assay of claim 12 where the peptide is DDDTSAVLQSGFRCGRGC.
44. The assay of claim 25 where the peptide is DDDTSAVLQSGFACGAGC.
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| US20110319293A1 (en) * | 2010-02-03 | 2011-12-29 | Butt Tauseef R | Synthetic protease substrates, assay methods using such substrates and kits for practicing the assay |
| US20170176272A1 (en) * | 2014-04-04 | 2017-06-22 | The Regents Of The University Of California | Plasmonic nanoparticle-based colorimetric stress memory sensor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20110319293A1 (en) * | 2010-02-03 | 2011-12-29 | Butt Tauseef R | Synthetic protease substrates, assay methods using such substrates and kits for practicing the assay |
| US20170176272A1 (en) * | 2014-04-04 | 2017-06-22 | The Regents Of The University Of California | Plasmonic nanoparticle-based colorimetric stress memory sensor |
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| KNELLER DANIEL W., PHILLIPS GWYNDALYN, WEISS KEVIN L., PANT SWATI, ZHANG QIU, O'NEILL HUGH M., COATES LEIGHTON, KOVALEVSKY ANDREY: "Unusual zwitterionic catalytic site of SARS–CoV-2 main protease revealed by neutron crystallography", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 295, no. 50, 1 December 2020 (2020-12-01), US , pages 17365 - 17373, XP055963386, ISSN: 0021-9258, DOI: 10.1074/jbc.AC120.016154 * |
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