POCT Detection of Pseudomonas aeruginosa by PGM and Application of Preventing Nosocomial Infection of Bronchoscopy

Background The primary pathogen responsible for bronchoscope contamination is Pseudomonas aeruginosa. Conventional techniques for bronchoscopy disinfection and pathogen identification methods are characterized by time-consuming and operation complexly. The objective of this research is to establish a prompt and precise method for the identification of Pseudomonas aeruginosa, with the ultimate goal of mitigating the risk of nosocomial infections linked to this pathogen. Methods The magnetic nanoparticles (MNPs) were synthesized in a single step, followed by the optimization of the coating process with antibodies and invertase to produce the bifunctionalized IMIc. Monoclonal antibodies were immobilized on microplates for the specific capture and enrichment of Pseudomonas aeruginosa. Upon the presence of Pseudomonas aeruginosa, the monoclonal antibodies, the test sample, and the IMIc formed sandwich structures. The subsequent addition of a sucrose solution allowed for the detection of glucose produced through invertase hydrolysis by a personal glucose meter, enabling quantitative assessment of Pseudomonas aeruginosa concentration. Results TEM image demonstrates that the MNPs exhibit a consistent spherical shape. NTA determined that the grain diameter of magnetic nanoparticles was 200 nm. FTIR spectrum revealed the successful modification of two carboxyl groups on the MNPs. The optimization of the incubation pH of the microplate-coated antibody was 7. The optimization of the incubation time of the microplate-coated antibody was 2 h. The optimization of the ligation pH for the polyclonal antibody was 5. Reaction times of polyclonal antibodies linked to magnetic beads was 1 h. The pH of invertase linked by magnetic beads was 4. Conclusion This article presents a novel qualitative and quantitative immunoassay for point-of-care monitoring of P. aeruginosa utilizing PGM as a readout. The PGM represents a convenient and accurate quantitative detection method suitable for potential clinical diagnostic applications.


Introduction
Pseudomonas aeruginosa, a Gram-negative pathogen, is frequently linked to nosocomial infections, infections in individuals with compromised immune systems, and persistent infections in patients diagnosed with structural lung disorders such as cystic fbrosis (CF).Pseudomonas aeruginosa is frequently implicated in nosocomial infections, presenting as pneumonia, surgical site infections, urinary tract infections, and bacteremia.Te prevalence of P. aeruginosa among all healthcare-associated infections is estimated to be between 7.1% and 7.3% [1,2].Pneumonia is the primary site of P. aeruginosa infection and is the most prevalent Gram-negative pathogen identifed in nosocomial pneumonia cases.Notably, the prevalence of P. aeruginosa infections has been steadily rising over the last ten years [3,4].Pseudomonas aeruginosa is responsible for a notably higher proportion of health medical-associated infections.A comprehensive observational point-prevalence study revealed that P. aeruginosa constituted 16.2% of infections among patients in intensive care units, with respiratory sources being the predominant site of P. aeruginosa infection [5].Healthcare-associated pneumonia (HAP) and ventilator-associated pneumonia (VAP) impose a substantial burden on the healthcare system, contributing to as much as 22% of all healthcare-acquired infections [6].
Endoscopes play a crucial role as diagnostic and therapeutic tools in contemporary medicine.Nonetheless, inadequate disinfection practices applied to endoscopes can potentially lead to the emergence of healthcare-acquired infections (HCAIs).Numerous instances have been documented wherein various types of endoscopes have been implicated in the occurrence of such outbreaks.Bronchoscopy is a widely utilized diagnostic and therapeutic modality in the feld, particularly within the domain of respiratory medicine.Bronchoscopy assumes a distinctive function in the identifcation and management of tracheobronchial lesions, pulmonary space-occupying pathologies, and various other ailments [7][8][9][10].Te utilization of electronic bronchoscopy is limited by its exorbitant cost and intricate design, necessitating multiple individuals to employ it repeatedly within a brief timeframe.Terefore, the cleaning and disinfection of the bronchoscope must be standardized to prevent cross-infection.
Bronchoscopes have been consistently identifed as the most commonly implicated type of endoscope in harboring pathogens [11,12].Given the potentially life-threatening consequences associated with Pseudomonas aeruginosa infections in patients, it is imperative to rigorously implement regular and meticulous cleaning protocols for bronchoscopes.Te conventional method for disinfection testing of bronchoscopes involves extracting a neutralizing sample solution from the biopsy orifce of the disinfected endoscope using a sterile syringe.Tis solution is then inoculated onto a sterile nutrient agar plate for culture and enumeration.However, this detection approach is characterized by a slow and time-consuming process.Consequently, there is an immediate requirement for the development of a rapid detection method for bronchoscopic pathogens.PGM has demonstrated a strong potential for application in rapid detection within various domains.However, previous research has predominantly focused on employing PGM for the detection of foodborne pathogenic microorganisms, primarily in the context of food safety.Conversely, the utilization of PGM for medical purposes, particularly within hospital settings, remains relatively limited, with a notable scarcity in the detection of common microbial pathogens.Te objective of this study is to develop a detection approach for internal quality control of nosocomial infection in the bronchoscopy room by leveraging the rapid, sensitive, and user-friendly outcomes provided by a glucose sensor.Tis method aims to prevent the incidence of aeruginosa-related nosocomial infections.

Te Immobilization of the Antibody on the Microplate.
As a frst step, the polystyrene microplate was noncovalently coated with anti-P.aeruginosa antibodies.Te microplate was incubated at 4 °C in a humid chamber for 2 h with a coating solution of anti-P.aeruginosa antibodies (PBS, pH 7.0).Te microplate was rinsed thoroughly and then incubated in 1% BSA for 1 hour to decrease nonspecifc binding.Afterwards, the microplate was repeatedly washed, and P. aeruginosa was detected.

Preparation of Invertase-MNPs-IgG Conjugates (IMIcs).
To produce invertase-MNPs-IgG conjugates (IMIcs), a onepot methodology was utilized to synthesize magnetic nanoparticles (MNPs) in accordance with the procedure outlined by Jia et al. [13].Typically, a solution was prepared by dissolving 0.68 mmol of trisodium citrate, 1.2 g of sodium acetate, and 4.0 mmol of FeCl3•6H 2 O in 20 mL of ethylene glycol.After heating and stirring to obtain a yellow turbid solution, it was transferred into an autoclave lined with Tefon and heated at 200 °C for 10 hours.T Subsequently, the precipitate was separated with a magnet and the magnetite nanoparticle was washed multiple times with ethanol and deionized water until the solution turned colorless and transparent, followed by drying at 80 °C under a vacuum.
MNPs were conjugated with invertase and detection antibodies.Following the dispersion of 2 mg of MNP in 500 µL of activation bufer (MEST, 10 mM, 0.05% Tween-20, pH 5.0), 2.5 mg and 2.5 mg of EDC and NHS were added, and the mixture was stirred for 30 min.Ten, the mixture was transferred to 500 µL of borate bufer (BB bufer, 20 mM, pH 5.0), and the products were washed twice and dispersed.MNP surface was frst incubated with 20 μg of anti-P.aeruginosa polyclonal antibody under stirring at 4 °C for 1 h.Ten, MNPs were incubated with 100 g of invertase for 1 hour at 4 °C in MES bufer (10 mM, pH 4.0).We washed and redispersed the obtained IMIc into 100 µL of storage solution (BBT, 5 mM, 0.05% Tween-20, 0.1% BSA, pH 7.2).

Establishment of Testing Procedures
. Figure 1 depicts the schematic representation of the immuno-PGM sensing system employed for the detection of target P. aeruginosa.Te system utilizes the anti-P.aeruginosa capture antibodycoated microplate.Initially, microtiter plates were loaded with 50 μL of P. aeruginosa standard or sample in pH 7.0 phosphate bufer and incubated at 37 °C for 30 minutes.

2
Journal of Analytical Methods in Chemistry Following the washing step, 50 μL of the previously prepared IMIc suspension was added to each well and incubated for 30 minutes at 37 °C.Subsequently, each well was injected with 50 μL of sucrose (0.5 g/mL) in pH 7 phosphate bufer after the plate was washed again.Finally, PGM was used to detect glucose concentration.

Validation of Clinical Samples.
We prepared a 10 mM glucose solution and measured it three times using a PGM.If the variation (CV) was ≤ 5%, PGM was said to have a good stability.Te Pseudomonas aeruginosa culture solution, with a concentration of 10 7 CFU/ml, was administered to the bronchoscope, followed by rinsing with 10 ml of normal saline.Te presence of Pseudomonas aeruginosa was subsequently assessed using a glucose sensor POCT.Te Pseudomonas aeruginosa culture solution, with a concentration of 107 CFU/ml, was administered onto the bronchoscope.Subsequently, employing this novel disinfection procedure, the disinfection duration was halved and then further reduced by three-quarters.Following disinfection, the bronchoscope was rinsed with 10 ml of normal saline.Te resulting rinsate was subjected to Pseudomonas aeruginosa content analysis using a glucose sensor pointof-care test (POCT).

Characterization of MNPs.
A transmission electron microscope (TEM) was utilized to examine the morphology of MNPs.Te TEM image demonstrates that the MNPs exhibit a consistent spherical shape, with an approximate diameter of 200 nm (Figure 2(a)).Nanoparticle tracking analysis (NTA) was utilized to determine the grain diameter of magnetic nanoparticles (MNPs).Our investigation revealed a singular peak exclusively at the magnitude of 200 nm.Nevertheless, the absence of peaks at alternative positions suggests that the synthesized nanomagnetic beads possess a consistent particle size, rendering them suitable for further investigations (Figure 2(b)).Moreover, the Fouriertransform infrared (FTIR) spectrum of unmodifed magnetic nanoparticles (MNPs) is depicted in Figure 2(c).Te FTIR peaks observed at 1617 cm −1 and 1385 cm −1 correspond to the asymmetric stretching mode and symmetric stretching mode of the COO-group, respectively.Tese fndings indicate the successful modifcation of carboxyl groups on the MNPs during the synthesis process, enabling direct one-step protein modifcation on the MNPs without the need for additional functionalization in subsequent experiments.

Optimization of Incubation pH and Incubation Time.
Initially, in order to apply the antibody onto the microplate, it is imperative to ascertain the suitable pH level of the PBS bufer.PBS bufer's pH range of gradients 4, 5, 6, 7, and 8 is considered.Subsequently, following the incubation period at 4 °C, the residual solution within the microplate was eliminated, and the protein concentration was assessed utilizing the BCA kit (Termo Fisher).In instances where the pH is suitable, the protein exhibits strong adherence to the microplate surface, resulting in a signifcantly reduced protein concentration.Conversely, when the pH is unsuitable, the protein connections are limited, leading to a substantial presence of antibodies in the liquid and consequently yielding a high measured protein concentration.It has been determined that the optimal pH value for this process is 7. Subsequently, the optimization of the reaction time for the microplate-linked antibody was conducted, with durations of 30 min, 1 h, 2 h, 3 h, and 4 h being tested.Te antibody was diluted using the PBS solution that exhibited the optimal pH value determined earlier, followed by the measurement of protein concentration.Te fndings indicated that the most favorable incubation efect was attained at the 2 h mark, with negligible alterations observed beyond this duration.Consequently, 2 h was designated as the selected reaction time (Figure 3).

Optimization of Bifunctional Magnetic Beads.
Te optimization of magnetic beads was conducted in two stages.First is the ligation of polyclonal antibodies using BB bufer.Te optimization of the ligation pH for the polyclonal antibody was conducted.Te pH level of BB utilized in the synthesis of invertase-MNPs-IgG conjugates (IMIcs) signifcantly impacts the coating efciency.As evidenced by the data presented in Table 1, negative results were obtained from the agglutination test at pH 3 and pH 7, and positive results were obtained at pH 4 and pH 6, respectively.A strong positive result was obtained at pH 5 (Table 1).Tese fndings suggest that the IgG in the antiserum was efectively coated onto the MNPs, and the conjugates exhibit a high capture efciency for P. aeruginosa.Second is the optimization of the ligation duration of the polyclonal antibody.Generally, the efcacy of IMIc is signifcantly infuenced by the quantity of IgG adhered to the MNPs.Te duration of incubation greatly impacts the rate at which proteins bind during the synthesis of IMIc.Te protein binding rate, as determined by the BCA kit, is depicted in Figure 4(a), where MNPs are saturated with antiserum and achieve a binding rate of 76.3% after 1 h.Consequently, 1 h was selected as the optimal incubation time condition.Lastly, the optimization of both the pH and time for the invertase ligation was performed.Due to the high cost and complexity associated with antibody preparation, we opted to employ unused  magnetic beads, devoid of antibody attachment, to efectively optimize the pH and ligation duration for invertase ligation, thereby avoiding the wastage of previously antibody-bound magnetic beads.Initially, invertase was immobilized onto magnetic beads at varying pH levels (3, 4, 5, 6, and 7), and the duration of immobilization was provisionally set at 1 h.Subsequently, the immobilized magnetic beads-invertase complex underwent two washes with PBS at pH 5. Following this, 20 microliters of the complex were introduced into a sucrose solution that had been previously prepared, and the resulting mixture was subjected to a reaction at 50 °C for a duration of 10 minutes.
Subsequently, 10 microliters of the resulting liquid were analyzed using a glucose sensor.Te labeling efciency of invertase on magnetic beads is directly infuenced by the pH of the MES bufer.Te results of our study indicated that the glucose sensor's signal exhibited an increase as pH decreased, reaching its maximum at a pH of 4. Our analysis further revealed that invertase displayed a positive charge when below or near its isoelectric point, leading to its attraction towards the negatively charged groups present on the surface of magnetic beads.Tis interaction facilitated the formation of chemical bonds.Consequently, a pH of 4 was chosen as the optimal incubation condition for linking glycozymes.Based on previous research, it is generally recommended to set the connection time between invertase and magnetic beads at 1 hour to achieve optimal connection.However, in this study, the magnetic beads were preconnected with polyclonal antibodies.To enhance the spatial structure connection between invertase and magnetic beads, the connection time of invertase was extended to 2 hours during the preparation of the "polyclonal antigenmagnetic beads-invertase" complex.Subsequently, the polyclonal antibody-magnetic beads-invertase complex was blocked with BSA solution, fxed in PBS, and stored for future use (Figure 4(b)).

Te Evaluation of Specifcity.
Te selectivity of PGM-POCT was assessed through the examination of 10 7 CFU/mL of P. aeruginosa and other microorganisms, including Escherichia coli, Klebsiellapneumoniae, Staphylococcus aureus, and yeast.As depicted in Figure 5(a), solely P. aeruginosa exhibited a positive reaction, whereas the remaining 4 non-P.aeruginosa strains displayed low PGM signal.Te consistent physical and chemical attributes of MNPs enhance the accuracy and precision of the detection analysis.Upon conjugation, the labeled samples exhibit favorable specifcity.Tese characteristics guarantee the PGM-POCT system's reliable reproducibility and selectivity throughout the experimental process.

Te Evaluation of Sensitivity.
In order to validate the efcacy of the PGM-POCT assay in targeting P. aeruginosa, a range of P. aeruginosa standards with varying concentrations were examined under optimal conditions on human P. aeruginosa antibody-coated polystyrene 96-well microplates.IMIcs were utilized as the signal-generation tags, and the readings were obtained using a glucometer.Figure 5(b) illustrates that the digital signals of the glucometer exhibit an increase corresponding to the rise in target P. aeruginosa levels in the sample.Tis observation aligns with the understanding that a higher concentration of P. aeruginosa results in the binding of a greater number of PGM-POCT through specifc antigen-antibody interactions.A strong linear correlation between the PGM signal (mM) and

Evaluation of the Actual Sample Test Efect.
In this study, we applied a P. aeruginosa culture medium directly onto the bronchoscope, followed by rinsing the bronchoscope with 10 ml of normal saline.Subsequently, the obtained sample was subjected to detection using our method, resulting in a quantifcation of 7.8 * 10 3 CFU/ml.In addition, we performed the smearing of P. aeruginosa, followed by normal disinfection, wherein the disinfection time was halved and then reduced by 3/4.Using this device, the results obtained were as follows: no detectable glucose concentration, no detectable glucose concentration, and 4.1 * 10 2 CFU/ml, respectively.

Discussion
Various types of electricity-based glucose sensors are available, with the GM sensor being the most commonly utilized due to its numerous advantages including speed, sensitivity, accuracy, and portability.In addition, the combination of PGM-POCT with nanomaterials has been shown to enhance sensitivity and accuracy, making it valuable for clinical diagnosis.As demonstrated by Wang et al., the utilization of PGM in conjunction with a microfuidic chip enabled the simultaneous detection of the three types of hepatitis B virus nucleic acids, resulting in an enhanced detection sensitivity [14].Te detection limit reached as low as 10 pM.Furthermore, PGM-POCT requires minimal equipment and personnel, further highlighting its practicality and efciency.Once the production of IMIc is consistently reliable, it presents a viable option for clinical application in resource-limited regions, particularly in the In this study, microplates coated with capture antibodies were employed to concentrate P. aeruginosa in bronchoscopic specimens.Te decision to utilize PGM-POCT for Pseudomonas aeruginosa was based on the practical clinical requirements.Within oncology hospitals, patients undergoing radiotherapy and chemotherapy are particularly vulnerable to infections and drug-resistant strains due to the immunosuppressive efects of these treatments.P. aeruginosa is a prevalent pathogen that frequently results in severe complications and mortality.In recent years, MNPs have gained popularity as nanomaterials in both basic research and clinical applications due to their advantageous characteristics, such as afordability, biocompatibility, and ease of use.MNPs have been modifed by coating them with antibodies and invertase to create a bifunctional IMIc, which has streamlined detection processes and reduced detection times.In clinical practice, prompt evaluation of disinfection quality following bronchoscopy procedures can efectively minimize patient wait times, given the high volume of daily procedures.Tis is of particular signifcance in nations such as China, characterized by a high volume of hospital patients.
Currently, the detection of P. aeruginosa primarily relies on colony culture methods and next-generation sequencing (NGS) techniques.While colony culture is cost-efective and straightforward, its turnaround time exceeds 48 hours, which is often inadequate for the timely needs of bronchoscopy procedures.NGS is advancing rapidly in China and is recognized as a critical tool for pathogen detection.However, NGS has yet to receive approval from Chinese medical regulatory authorities (NMPA), and its cost remains above $500, imposing signifcant economic strain on patients.In contrast, PGM-POCTofers high sensitivity, ease of use, and objective results, with a single test costing less than $30.Nevertheless, PGM-POCT is still in the experimental development phase.Future research may focus on incorporating assembly line production and freeze-drying techniques for processing IMIc, which could substantially reduce both operational and storage costs.Tis approach represents a promising direction for subsequent research eforts.

Conclusion
Tis article presents a novel qualitative and quantitative immunoassay for point-of-care monitoring of P. aeruginosa utilizing PGM as a readout.In addition, biomolecules can be efciently labeled and separated using MNPs in conjunction with a permanent magnet.Te protein-modifed MNPs demonstrate a strong selectivity and resistance to interference.Consequently, the newly developed PGM-ICA employing IMIc represents a promising and versatile approach for detecting P. aeruginosa, with potential applicability to other bacterial species through modifcation of the antibody.

Figure 2 :
Figure 2: Characterization of MNPs.(a) TEM image demonstrates that the MNPs exhibit a consistent spherical shape.(b) NTA determined that the grain diameter of magnetic nanoparticles was 200 nm.(c) FTIR spectrum revealed the successful modifcation of two carboxyl groups on the MNPs.

Figure 3 :
Figure 3: Optimization of the experimental conditions.(a) Optimization of incubation pH.(b) Optimization of incubation time.

Figure 4 :Figure 5 :
Figure 4: Optimization of bifunctional magnetic beads.(a) Reaction times of polyclonal antibodies linked to magnetic beads.(b) pH of invertase linked by magnetic beads.