Functionalized Hydrogel Pellets Combined With Colorimetric/Pressure Sensing For Point-of-care Testing Of Pathogenic Bacteria

Infectious diseases caused by pathogenic bacteria have been increasingly serious threats to public health.Thus,developing rapid,accurate,and sensitive methods for detecting and identifying pathogens is important.As for the ASSURED criteria from WHO,an analysis method should be advocated to be affordable,sensitive,specific,user-friendly,rapid and robust,equipment-free,and deliverable to end users.Point-of-care testing（POCT）approach offers the possibility of providing rapid diagnostic results with portable,economical,and user-friendly devices.Colorimetric analysis is a convenient method in POCT that enables qualitative or semi-quantitative detection of target analytes through visual observation of color changes.Due to its simplicity and visualization without the need for complex instruments,colorimetric analysis plays a crucial role in the detection of pathogenic bacteria.To improve its sensitivity,the development of some specific probes and signal amplification strategies are needed to emphasize color changes.In our daily life,some classical physical principles are widely applied in visually quantitative devices,such as thermometers,barometers,and hygrometers.Such devices are ideal models for POCT,as they provide simple,portable,sensitive,and quantitative detection without any external instrument or power source.Pressure,a simple physical signal,can be measured by a digital pressure meter.Gas-generation reactions,which can produce considerable amounts of gas,can lead to obvious pressure increases in a closed container and have a signal amplification effect.Therefore,the design of a POCT strategy based on catalytic gas production reaction for the detection of pathogenic bacteria will have extremely high analytical sensitivity.Hydrogel is a kind of three-dimensional（3D）polymeric network consisting of hydrophilic natural or synthetic polymers.It has been applied in the fields of biomedicine and biosensing,such as wound dressing,tissue engineering,drug delivery,and disease diagnostics.Hydrogel has a significant advantage as a biomimetic 3D matrix,which provides an ideal environment for recognition molecules in biosensors,such as antibodies,aptamers,and enzymes.This provides ideal reaction conditions for recognition molecules to interact with their targets within the aqueous environment of the hydrogel.Therefore,the hydrogel can be used as a carrier for recognition molecules to capture and detect various analytes.Inspired by the knowledge described above,herein,different molecular-functionalized hydrogel pellets were used as target capture and separation tools,and functionalized platinum nanomaterials including platinum-coated palladium core-frame dendrites（Pd@Pt NDs）,platinum-coated gold nanoparticles（Au@Pt NPs）,and platinum-loaded metal-organic framework（PCN-222（Pt））were used as signal tracers.The strategies of POCT based on molecular recognition coupled with colorimetric and pressure sensing were developed for quantitative detection of Staphylococcus aureus（S.aureus）,Escherichia coli O157:H7（E.coli O157:H7）and Pseudomonas aeruginosa（P.aeruginosa）.The contents are described in detail as follows:1.Selection and application of E.coli aptamersThe aptamer can recognize a variety of targets specifically or non-specifically.Therefore,aptamers are usually used in biosensor technology to detect the target qualitatively or quantitatively.Herein,two different aptamers were selected and validated for specific recognition E.coli ATCC25922 and E.coli O157:H7,respectively.Pd@Pt NDs with excellent peroxidase-like activity were successfully synthesized and can catalyze the obvious color reaction of 3,3’,5,5’-tetramethylbenzidine（TMB）.A catalytic colorimetric biosensing platform was constructed for the detection of E.coli O157:H7 using aptamers as the recognition molecules,and Pd@Pt NDs as the signal tracers.The correlation between the concentration of E.coli O157:H7 and the absorbance signal was found to be linear from 3.8×10³ to 3.8×10⁷ cfu/m L with a detection limit of 6.3×10² cfu/m L.This method was successfully applied for the detection of E.coli O157:H7 in real samples,such as 0.9%Na Cl,lake water,and milk,with recoveries from 84.2%to 115.8%and a relative standard deviation（RSD）of less than 9.7%.The platform provides a simple and efficient method for the detection of pathogenic bacteria.2.Shape-encoded functional hydrogel pellets for multiplexed detection of pathogenic bacteria using a gas pressure sensorAs a member of the lantibiotics family,nisin is a bacteriocin produced by Lactococcus lactis.It binds to the lipid II unit in the cell membrane of Gram-positive bacteria.Thus,nisin with binding ability can be potentially applied as a recognition molecule for Gram-positive bacteria.Among various lectins,concanavalin A（Con A）is a mannose-and glucose-binding protein extracted from Canavalia ensiformis.It is a promising recognition molecule for Gram-negative bacteria because it can specifically bind to bacterial surface O-antigen.A gas pressure sensor was developed for multiplexed detection of S.aureus and E.coli O157:H7 with shape-encoded functional hydrogel pellets.Nisin-and Con A-functionalized hydrogel pellets（NFH pellets and CFH pellets）were prepared into spherical and square shapes for recognizing S.aureus and E.coli O157:H7,respectively.NFH pellets were used to capture S.aureus from a sample matrix utilizing the interaction between nisin and lipid II unit in its cell membrane,and Au@Pt NPs-modified aptamer 1 was used as a signal tracer.Similarly,CFH pellets and Au@Pt NPs-modified aptamer 2 were adopted for capturing and tracing E.coli O157:H7,respectively.Thus,the sandwich complexes of Nisin-S aureus-aptamer1 and Con A-E.coli O157:H7-aptamer 2 were formed with a double-molecule recognition pattern.By using the shape-encoded functional hydrogel pellets,the two pathogenic bacteria were discriminated and detected in the closed containers by pressure signals from the Au@Pt NPs catalyzed gas generation reaction.3.Isolation and purification of P.aeruginosa phageP.aeruginosa,a Gram-negative opportunistic pathogen,is ubiquitous in the environment and can cause various infectious diseases.Therefore,the development of rapid and accurate methods for detecting P.aeruginosa is of great importance.Herein,P.aeruginosa phage v B＿Pae P-JZ1（JZ1）was isolated from river water,and the potential application of JZ1 in the detection of P.aeruginosa was investigated by studying its biological properties.The results showed that phage JZ1 was a lytic phage and belonged to the family Podoviridae.In a liquid-rich medium,phage titer can be amplified to～10¹³ pfu/m L.The optimal multiplicity of infection（MOI）of phage JZ1 infecting P.aeruginosa was determined to be 1.The temperature and p H stability test results indicated that the phage exhibits optimal survival temperatures ranging from 20 to 50℃and optimal p H levels between 6 and 11.The successful isolation of the phage JZ1provides a new recognition molecule for achieving specific detection of P.aeruginosa.4.A 3D printed“both-in-one”device assembled by hydrogel modified with phages for POCT of P.aeruginosaHerein,a 3D printed device combined with a hydrogel pellet modified with phages was designed for POCT of P.aeruginosa with both colorimetric and pressure readout modes.Due to its host specificity,phage JZ1 was used as a recognizing agent for modifying the hydrogel pellet,and the modified hydrogel pellet was assembled into the3D printed device to act as the sensing interface.Polymyxin B（PMB）was tagged with Pd@Pt NDs with excellent peroxidase-like activity to act as the colorimetric and pressure signal tracer.P.aeruginosa can be quantified within the concentration ranges of 2.6×10³ cfu/m L to 2.6×10⁸ cfu/m L and 2.6×10² cfu/m L to 2.6×10⁷ cfu/m L with colorimetric and pressure readout modes,respectively.The both modes can achieve quantitation of P.aeruginosa within 25 min.Thus the“both-in-one”3D printed device with dual-mode readout function offers a rapid,sensitive,and specific platform for POCT of pathogenic bacteria.5.A pressure sensor for the analysis of P.aeruginosa based on the metal-organic framework PCN-222（Pt）PCN-222（Pt）was successfully prepared by utilizing the metal-organic framework PCN-222 as a carrier for Pt atoms,which was subjected to a comprehensive set of characterizations.In comparison to Pd@Pt NDs and Au@Pt NPs,the prepared PCN-222（Pt）demonstrated superior peroxidase-like activity with higher efficiency in catalyzing the decomposition of H₂O₂to produce O₂.PMB was tagged with PCN-222（Pt）to act as the pressure signal tracer,while P.aeruginosa phage JZ1 was used as a recognizing agent for modifying the hydrogel pellets.The quantitative detection of P.aeruginosa was achieved by utilizing pressure signals.The signal amplification strategy based on PCN-222（Pt）-catalyzed gas generation significantly enhances the detection sensitivity of P.aeruginosa.This study presents a novel signal output probe for the applications of pressure sensing in the detection of pathogenic bacteria.6.3D printed analysis device for POCT of P.aeruginosaA colorimetric analysis device integrated with“incubation-washing-detection”was prepared using 3D printed transparent resin for POCT of P.aeruginosa.P.aeruginosa phage JZ1 was used as the recognition reagent for modifying magnetic beads（MBs）.Meanwhile,PCN-222（Pt）was labeled with PMB and utilized as a signal tracer.In the presence of H₂O₂,PCN-222（Pt）catalyzes the colorimetric reaction of TMB,which enables quantitative analysis of P.aeruginosa using colorimetric signals.The prepared analysis device consists of three functional wells,where MBs can be guided to different wells through an external magnetic field for relevant operations.The combination of a3D-printed integrated device and magnetic MBs separation avoids repeated magnetic separation and reduces the detection time.Furthermore,the 3D device can be directly transferred to a 96-well plate holder for colorimetric measurements,which significantly improves the detection efficiency.This study provides a new method for the development of POCT for pathogenic bacteria.In summary,this study selected two aptamers and isolated a phage for the recognition of S.aureus,E.coli O157:H7,and P.aeruginosa,respectively.Hydrogel pellets are utilized as carriers for immobilizing recognition molecules to capture and separate target analytes.Three types of synthesized nanomaterials,Pd@Pt NDs,Au@Pt NPs,and PCN-222（Pt）,all exhibited significant peroxidase-like activity,which can catalyze a visible color reaction of TMB and catalyze the decomposition of H₂O₂ to produce a large amount of O₂.These nanomaterials were designed as signal probes for colorimetric/pressure sensing strategies,respectively.New methods for POCT of the above three pathogenic bacteria were established based on molecular recognition coupled with colorimetric/pressure sensing with the advantages of high sensitivity,rapid analysis,and simple operation.It is of great significance for environmental monitoring,food safety,healthcare,and biomedical diagnostics.