Label-free integrated microfluidic plasmonic biosensor from vertical-cavity surface-emitting lasers for SARS-CoV-2 receptor binding domain protein detection.

The nanoplasmonic sensor of the nanograting array has a remarkable ability in label-free and rapid biological detection. The integration of the nanograting array with the standard vertical-cavity surface-emitting lasers (VCSEL) platform can achieve a compact and powerful solution to provide on-chip light sources for biosensing applications. Here, a high sensitivity and label-free integrated VCSELs sensor was developed as a suitable analysis technique for COVID-19 specific receptor binding domain (RBD) protein. The gold nanograting array is integrated on VCSELs to realize the integrated microfluidic plasmonic biosensor of on-chip biosensing. The 850 nm VCSELs are used as a light source to excite the localized surface plasmon resonance (LSPR) effect of the gold nanograting array to detect the concentration of attachments. The refractive index sensitivity of the sensor is 2.99 × 106 nW/RIU. The aptamer of RBD was modified on the surface of the gold nanograting to detect the RBD protein successfully. The biosensor has high sensitivity and a wide detection range of 0.50 ng/mL - 50 µg/mL. This VCSELs biosensor provides an integrated, portable, and miniaturized idea for biomarker detection.

Effect of near-infrared and blue laser light on vero E6 cells SARS-CoV-2 infection model.

Photobiomodulation therapy (PBMT) employing laser light has been emerging as a safe strategy to challenge viruses. In this study the effect of blue and near-infrared (NIR) laser light was assessed in an in vitro model of SARS-CoV-2 infection. PBMT at blue wavelength inhibited viral amplification when the virus was directly irradiated and then transferred to cell culture and when cells already infected were treated. The NIR wavelength resulted less efficacious showing a minor effect on the reduction of the viral load. The cells receiving the irradiated virus or directly irradiated rescued their viability to level comparable to not treated cells. Virion integrity and antigenicity were preserved after blue and NIR irradiation, suggesting that the PBMT antiviral effect was not correlated to viral lipidic envelope disruption. Our results suggested that PBMT can be considered a valid strategy to counteract SARS-CoV-2 infection, at least in vitro.

Breath analysis by ultra-sensitive broadband laser spectroscopy detects SARS-CoV-2 infection.

Rapid testing is essential to fighting pandemics such as coronavirus disease 2019 (COVID-19), the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Exhaled human breath contains multiple volatile molecules providing powerful potential for non-invasive diagnosis of diverse medical conditions. We investigated breath detection of SARS-CoV-2 infection using cavity-enhanced direct frequency comb spectroscopy (CE-DFCS), a state-of-the-art laser spectroscopic technique capable of a real-time massive collection of broadband molecular absorption features at ro-vibrational quantum state resolution and at parts-per-trillion volume detection sensitivity. Using a total of 170 individual breath samples (83 positive and 87 negative with SARS-CoV-2 based on reverse transcription polymerase chain reaction tests), we report excellent discrimination capability for SARS-CoV-2 infection with an area under the receiver-operating-characteristics curve of 0.849(4). Our results support the development of CE-DFCS as an alternative, rapid, non-invasive test for COVID-19 and highlight its remarkable potential for optical diagnoses of diverse biological conditions and disease states.

Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser.

NendoU from SARS-CoV-2 is responsible for the virus's ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.

The effect of systemic versus local transcutaneous laser therapy on tension-type cephalea and orofacial pain in post-COVID-19 patients: A pragmatic randomized clinical trial.

INTRODUCTION: Orofacial pain and tensional cephalea were symptoms commonly reported in COVID-19 patients, even after recovery, and were considered chronic pain in these cases. The aim of this research is to evaluate the effect of the application of photobiomodulation with red and infrared lasers applied locally and systemically. METHODS AND ANALYSIS: For this purpose, individuals who have been diagnosed with COVID-19 and have had a tension headache and/or orofacial pain for more than 3 months will be selected by convenience. The participants will be divided into two different groups: G1-photobiomodulation with red and infrared laser with local application on the pain points (808 nm and 660 nm, 100 mW, 6 J per point) and G2-photobiomodulation with red laser with transcutaneous application on the radial artery (660 nm, 100 mW, 30 minutes). All participants will be treated for a period of 4 weeks, with 8 application sessions. The effects will be measured by means of blood lactate level, Brief Pain Inventory, Visual Analog Scale (VAS), and Cephalea Impact Test. The data will be collected weekly before and after the treatment, and the following tests will be applied: Analysis of variance (ANOVA), Tukey paired t test, Kruskal-Wallis, or Wilcoxon, according to data distribution. α = 0.05 will be considered as the level of statistical significance. ETHICS AND DISSEMINATION: This study was approved by the Research Projects Committee of the Nove de Julho University (approval number 4.673.963). Results will be disseminated through peer-reviewed journals and events for the scientific and clinical community, and the general public. It is registered in the ClinicalTrials.gov database with the number NCT05430776.

Ultrafast inactivation of SARS-CoV-2 with 266 nm lasers.

Disinfection eliminates pathogenic microorganisms and ensures a biosafe environment for human beings. The rapid spread of COVID-19 is challenging traditional disinfection methods in terms of reducing harmful side effects and conducting faster processes. Spraying large-scale chemical disinfectants is harmful to individuals and the environment, while UV lamp and light-emitting diode (LED) disinfection still requires a long exposure time due to the low irradiance and highly divergent beam characteristics. Given that a laser maintains a high irradiance over a long distance, we studied the effectiveness of lasers as a new disinfection method, and the results show the capability for ultrafast inactivation of SARS-CoV-2 virus with a 266 nm laser. This work confirms UV lasers as a good candidate for disinfection.

Visualizing endoscopy-generated aerosols with laser light scattering (with videos).

BACKGROUND AND AIMS: Upper GI endoscopy is speculated to be an aerosol-generating procedure (AGP). Robust evidence exists for aerosol transmission of severe acute respiratory syndrome coronavirus 2. The quality of data available confirming aerosol generation during GI endoscopy is limited. We aimed to objectively demonstrate that GI endoscopy is an AGP and illustrate the mechanism by which the greatest risk for aerosolization of droplets during endoscopy may occur. METHODS: Aerosolized droplets generated during insertion and withdrawal of an endoscope and with passage of various tools through the endoscopic working channel using 2 experimental apparatuses modeling an upper GI tract (ie, a fluid-filled tube and a lamb esophagus) were qualitatively assessed by laser light scattering. RESULTS: Insertion and withdrawal of the upper endoscope into the upper GI tract models generated numerous aerosolized particles. A large number of brightly scattering particles were observed at the site of insertion and withdrawal of the endoscope. Passage of a cytology brush, biopsy forceps, and hemostatic clip through the working endoscope channel also generated aerosolized particles but in fewer numbers. There was no significant variation in quantity or brightness of droplets generated on testing different biopsy valve cap models or when suctioning fluid with an open versus closed biopsy valve cap. These results were reproducible over several trials. CONCLUSIONS: We illustrate in an objective manner that upper GI endoscopy is an AGP. These findings may have implications for transmission of infectious airborne pathogens outside of severe acute respiratory syndrome coronavirus 2 and can help to inform guidance on appropriate personal protective equipment use and other measures for transmission risk mitigation during GI endoscopy.

Electrolyte Analysis in Blood Serum by Laser-Induced Breakdown Spectroscopy Using a Portable Laser.

The fast and reliable analysis of electrolytes such as K, Na, Ca in human blood serum has become an indispensable tool for diagnosing and preventing diseases. Laser-induced breakdown spectroscopy (LIBS) has been demonstrated as a powerful analytical technique on elements. To apply LIBS to the quantitative analysis of electrolyte elements in real time, a self-developed portable laser was used to measure blood serum samples supported by glass slides and filter paper in this work. The partial least squares regression (PLSR) method was employed for predicting the concentrations of K, Na, Ca from serum LIBS spectra. Great prediction accuracies with excellent linearity were obtained for the serum samples, both on glass slides and filter paper. For blood serum on glass slides, the prediction accuracies for K, Na, Ca were 1.45%, 0.61% and 3.80%. Moreover, for blood serum on filter paper, the corresponding prediction accuracies were 7.47%, 1.56% and 0.52%. The results show that LIBS using a portable laser with the assistance of PLSR can be used for accurate quantitative analysis of elements in blood serum in real time. This work reveals that the handheld LIBS instruments will be an excellent tool for real-time clinical practice.

Ultrafast analysis of peptides by laser diode thermal desorption-triple quadrupole mass spectrometry.

RATIONALE: The COVID-19 pandemic demonstrated the importance of high-throughput analysis for public health. Given the importance of surface viral proteins for interactions with healthy tissue, they are targets of interest for mass spectrometry-based analysis. For that reason, the possibility of detecting and quantifying peptides using a high-throughput technique, laser diode thermal desorption-triple quadrupole mass spectrometry (LDTD-QqQMS), was explored. METHODS: Two peptides used as models for small peptides (leu-enkephalin and endomorphin-2) and four tryptic peptides (GVYYPDK, NIDGYFK, IADYNYK, and QIAPGQTGK) specific to the SARS-CoV-2 Spike protein were employed. Target peptides were analyzed individually in the positive mode by LDTD-QqQMS. Peptides were quantified by internal calibration using selected reaction monitoring transitions in pure solvents and in samples spiked with 20 µg mL-1 of a bovine serum albumin tryptic digest to represent real analysis conditions. RESULTS: Low-energy fragment ions (b and y ions) as well as high-energy fragment ions (c and x ions) and some of their corresponding water or ammonia losses were detected in the full mass spectra. Only for the smallest peptides, leu-enkephalin and endomorphin-2, were [M + H]+ ions observed. Product ion spectra confirmed that, with the experimental conditions used in the present study, LDTD transfers a considerable amount of energy to the target peptides. Quantitative analysis showed that it was possible to quantify peptides using LDTD-QqQMS with acceptable calibration curve linearity (R2 > 0.99), precision (RSD < 18.2%), and trueness (bias < 8.3%). CONCLUSIONS: This study demonstrated for the first time that linear peptides can be qualitatively and quantitatively analyzed using LDTD-QqQMS. Limits of quantification and dynamic ranges are still inadequate for clinical applications, but other applications where higher levels of proteins must be detected could be possible with LDTD. Given the high-throughput capabilities of LDTD-QqQMS (>15 000 samples in less than 43 h), more studies are needed to improve the sensitivity for peptide analysis of this technique.

Freestanding Laser-Induced Graphene Ultrasensitive Resonative Viral Sensors.

Early and reliable detection of an infectious viral disease is critical to accurately monitor outbreaks and to provide individuals and health care professionals the opportunity to treat patients at the early stages of a disease. The accuracy of such information is essential to define appropriate actions to protect the population and to reduce the likelihood of a possible pandemic. Here, we show the fabrication of freestanding laser-induced graphene (FLIG) flakes that are highly sensitive sensors for high-fidelity viral detection. As a case study, we show the detection of SARS-CoV-2 spike proteins. FLIG flakes are nonembedded porous graphene foams ca. 30 µm thick that are generated using laser irradiation of polyimide and can be fabricated in seconds at a low cost. Larger pieces of FLIG were cut forming a cantilever, used as suspended resonators, and characterized for their electromechanics behavior. Thermomechanical analysis showed FLIG stiffness comparable to other porous materials such as boron nitride foam, and electrostatic excitation showed amplification of the vibrations at frequencies in the range of several kilo-hertz. We developed a protocol for aqueous biological sensing by characterizing the wetting dynamic response of the sensor in buffer solution and in water, and devices functionalized with COVID-19 antibodies specifically detected SARS-CoV-2 spike protein binding, while not detecting other viruses such as MS2. The FLIG sensors showed a clear mass-dependent frequency response shift of ∼1 Hz/pg, and low nanomolar concentrations could be detected. Ultimately, the sensors demonstrated an outstanding limit of detection of 2.63 pg, which is equivalent to as few as ∼5000 SARS-CoV-2 viruses. Thus, the FLIG platform technology can be utilized to develop portable and highly accurate sensors, including biological applications where the fast and reliable protein or infectious particle detection is critical.

Saving 80% Polypropylene in Facemasks by Laser-Assisted Melt-Blown Nanofibers.

The ongoing coronavirus (COVID-19) pandemic requires enormous production of facemasks and related personal protection materials, thereby increasing the amount of nondegradable plastic waste. The core material for facemasks is melt-blown polypropylene (PP) fiber. Each disposable facemask consumes ∼0.7 g of PP fibers, resulting in annual global consumption and disposal of more than 1 150 000 tons of PP fibers annually. Herein, we developed a laser-assisted melt-blown (LAMB) technique to manufacture PP nanofibers with a quality factor of 0.17 Pa-1 and significantly reduced the filter's weight. We demonstrated that a standard surgical facemask could be made with only 0.13 g of PP nanofibers, saving approximately 80% of the PP materials used in commercial facemasks. Theoretical analysis and modeling were also conducted to understand the LAMB process. Importantly, nanofibers can be easily scaled up for mass production by upgrading traditional melt blown line with scanning laser-assisted melt-blown (SLAMB).

Label-free laser spectroscopy for respiratory virus detection: A review.

Infectious diseases are among the most severe threats to modern society. Current methods of virus infection detection based on genome tests need reagents and specialized laboratories. The desired characteristics of new virus detection methods are noninvasiveness, simplicity of implementation, real-time, low cost and label-free detection. There are two groups of methods for molecular biomarkers' detection and analysis: (i) a sample physical separation into individual molecular components and their identification, and (ii) sample content analysis by laser spectroscopy. Variations in the spectral data are typically minor. It requires the use of sophisticated analytical methods like machine learning. This review examines the current technological level of laser spectroscopy and machine learning methods in applications for virus infection detection.

All-in-One Optofluidic Chip for Molecular Biosensing Assays.

Integrated biosensor platforms have become subjects of high interest for consolidated assay preparation and analysis to reduce sample-to-answer response times. By compactly combining as many biosensor processes and functions as possible into a single lab-on-chip device, all-in-one point-of-care devices can aid in the accessibility and speed of deployment due to their compact size and portability. Biomarker assay preparation and sensing are functionalities that are often carried out on separate devices, thus increasing opportunity of contamination, loss of sample volume, and other forms of error. Here, we demonstrate a complete lab-on-chip system combining sample preparation, on-chip optofluidic dye laser, and optical detection. We first show the integration of an on-chip distributed feedback dye laser for alignment-free optical excitation of particles moving through a fluidic channel. This capability is demonstrated by using Rhodamine 6G as the gain medium to excite single fluorescent microspheres at 575 nm. Next, we present an optofluidic PDMS platform combining a microvalve network (automaton) for sample preparation of nanoliter volumes, on-chip distributed feedback dye laser for target excitation, and optical detection. We conduct concurrent capture and fluorescence tagging of Zika virus nucleic acid on magnetic beads in 30 min. Target-carrying beads are then optically excited using the on-chip laser as they flow through an analysis channel, followed by highly specific fluorescence detection. This demonstration of a complete all-in-one biosensor is a tangible step in the development of a rapid, point-of-care device that can assist in limiting the severity of future outbreaks.

Highly sensitive aptasensor for the detection of SARS-CoV-2-RBD using aptamer-gated methylene blue@mesoporous silica film/laser engraved graphene electrode.

Herein, an aptasensor was designed to detect the receptor-binding domain of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2-RBD) based on the encapsulation of the methylene blue (MB) inside the mesoporous silica film (MPSF), and an aptamer as an electrochemical probe, a porous matrix, and a bio-gatekeeper, respectively. The signal analysis of the proposed aptasensor indicated that the surface coverage of the encapsulated MB inside the MPSF (MB@MPSF) was 1.9 nmol/cm2. Aptamers were capped the MB@MPSF, avoiding the release of MB into the solution via the electrostatic attraction between the positively charged amino groups of the MPSF and negatively charged phosphate groups of the aptamers. Therefore, the electrochemical signal of the encapsulated MB in the absence of the SARS-CoV-2-RBD was high. In the presence of SARS-CoV-2-RBD, the aptamers that had a high affinity to the SARS-CoV-2-RBD molecules were removed from the electrode surface to interact with SARS-CoV-2-RBD. It gave rise to the release of the MB from the MPSF to the solution and washed away on the electrode surface. Therefore, the electrochemical signal of the aptasensor decreased. The electrochemical signal was recorded with a square wave voltammetry technical in the range of 0.5-250 ng/mL of SARS-CoV-2-RBD in a saliva sample. The limit of detection was found to be 0.36 ng/mL. Furthermore, the selectivity factor values of the proposed aptasensor to 32 ng/mL SARS-CoV-2-RBD in the presence of C-reactive protein, hemagglutinin, and neuraminidase of influenza A virus were 35.9, 11.7, and 17.37, respectively, indicating the high selectivity of the proposed aptasensor.

A defined road to tracheal reconstruction: laser structuring and cell support for rapid clinic translation.

One of the severe complications occurring because of the patient's intubation is tracheal stenosis. Its incidence has significantly risen because of the COVID-19 pandemic and tends only to increase. Here, we propose an alternative to the donor trachea and synthetic prostheses-the tracheal equivalent. To form it, we applied the donor trachea samples, which were decellularized, cross-linked, and treated with laser to make wells on their surface, and inoculated them with human gingiva-derived mesenchymal stromal cells. The fabricated construct was assessed in vivo using nude (immunodeficient), immunosuppressed, and normal mice and rabbits. In comparison with the matrix ones, the tracheal equivalent samples demonstrated the thinning of the capsule, the significant vessel ingrowth into surrounding tissues, and the increase in the submucosa resorption. The developed construct was shown to be highly biocompatible and efficient in trachea restoration. These results can facilitate its clinical translation and be a base to design clinical trials.

Ultrafast-UV laser integrating cavity device for inactivation of SARS-CoV-2 and other viruses.

Ultraviolet (UV) irradiation-based methods used for viral inactivation have provided an important avenue targeting severe acute respiratory-syndrome coronavirus-2 (SARS-CoV-2) virus. A major problem with state-of-the-art UV inactivation technology is that it is based on UV lamps, which have limited efficiency, require high power, large doses, and long irradiation times. These drawbacks limit the use of UV lamps in air filtering systems and other applications. To address these limitations, herein we report on the fabrication of a device comprising a pulsed nanosecond 266 nm UV laser coupled to an integrating cavity (LIC) composed of a UV reflective material, polytetrafluoroethylene. Previous UV lamp inactivation cavities were based on polished walls with specular reflections, but the diffuse reflective UV ICs were not thoroughly explored for virus inactivation. Our results show that LIC device can inactivate several respiratory viruses including SARS-CoV-2, at ~ 1 ms effective irradiation time, with > 2 orders of magnitude higher efficiency compared to UV lamps. The demonstrated 3 orders of magnitude cavity enhancement relative to direct exposure is crucial for the development of efficient real-time UV air and water purification systems. To the best of our knowledge this is the first demonstration of LIC application for broad viral inactivation with high efficiency.

Inactivation of SARS-CoV-2 by a chitosan/α-Ag2WO4 composite generated by femtosecond laser irradiation.

In the current COVID-19 pandemic, the next generation of innovative materials with enhanced anti-SARS-CoV-2 activity is urgently needed to prevent the spread of this virus within the community. Herein, we report the synthesis of chitosan/α-Ag2WO4 composites synthetized by femtosecond laser irradiation. The antimicrobial activity against Escherichia coli, Methicilin-susceptible Staphylococcus aureus (MSSA), and Candida albicans was determined by estimating the minimum inhibitory concentration (MIC) and minimal bactericidal/fungicidal concentration (MBC/MFC). To assess the biocompatibility of chitosan/α-Ag2WO4 composites in a range involving MIC and MBC/MFC on keratinocytes cells (NOK-si), an alamarBlue™ assay and an MTT assay were carried out. The SARS-CoV-2 virucidal effects was analyzed in Vero E6 cells through viral titer quantified in cell culture supernatant by PFU/mL assay. Our results showed a very similar antimicrobial activity of chitosan/α-Ag2WO4 3.3 and 6.6, with the last one demonstrating a slightly better action against MSSA. The chitosan/α-Ag2WO4 9.9 showed a wide range of antimicrobial activity (0.49-31.25 µg/mL). The cytotoxicity outcomes by alamarBlue™ revealed that the concentrations of interest (MIC and MBC/MFC) were considered non-cytotoxic to all composites after 72 h of exposure. The Chitosan/α-Ag2WO4 (CS6.6/α-Ag2WO4) composite reduced the SARS-CoV-2 viral titer quantification up to 80% of the controls. Then, our results suggest that these composites are highly efficient materials to kill bacteria (Escherichia coli, Methicillin-susceptible Staphylococcus aureus, and the yeast strain Candida albicans), in addition to inactivating SARS-CoV-2 by contact, through ROS production.

Traveling tunable laser projector for UV-blue disinfection dose determinations.

As the COVID-19 pandemic was overtaking the world in the spring of 2020, the National Institute of Standards and Technology (NIST) began collaborating with the National Biodefense Analysis and Countermeasures Center to study the inactivation of SARS-CoV-2 after exposure to different ultraviolet (UV) and blue light wavelengths. This paper describes a 1 kHz pulsed laser and projection system used to study the doses required to inactive SARS-CoV-2 over the wavelength range of 222 to 488 nm. This paper builds on NIST's previous work for water pathogen inactivation using UV laser irradiation. The design of the laser and projection system and its performance in a Biosafety Level 3 (BSL-3) laboratory are given. The SARS-CoV-2 inactivation results (published elsewhere by Schuit, M.A., et al., expected 2022) demonstrate that a tunable laser projection system is an invaluable tool for this research.

Lasting Effects: Seven Year Results of the Castrop Nomogram for Femtosecond Laser-Assisted Paired Corneal Arcuate Incisions.

PURPOSE: Long-term results of arcuate incisions are rarely reported. This is unfortunate as long-term stability of astigmatic correction is of great interest to surgeons performing astigmatic correction. This study investigates the 7 year stability of results after application of femtosecond laser-assisted arcuate incisions with the Castrop nomogram. METHODS: Prospective interventional case series at the Augen- und Laserklinik, Castrop-Rauxel, Germany. Single site, single surgeon study. Seven year results of cataract patients with low to moderate corneal astigmatism receiving femtosecond laser-assisted arcuate incisions using a TechnolasVictus SW 2.7 (Bausch & Lomb Inc, Dornach, Germany) were assessed and compared to 1 year results. Outcome evaluation was based on astigmatic vector analysis, manifest refraction, and visual acuity. RESULTS: The study analyzed 19 eyes of 19 patients 7 years after surgery. Ocular residual astigmatism changed from -0.26 to -0.39 D. Preoperative corneal astigmatism was -1.51 D. Correction Index changed from 1.0 to 1.16. The magnitude of difference vector changed from 0.26 to 0.39 D. The index of success changed from 0.20 to 0.29. Spherical equivalent remained stable. A slight tendency to change toward astigmatic overcorrection was mainly observed for patients with preoperative with the rule astigmatism, but not with patients with against the rule astigmatism. CONCLUSIONS: The Castrop nomogram showed stable results 7 years after surgery. Similar to toric IOL surgery, it is advisable to be less aggressive when correcting with the rule astigmatism, to avoid overcorrection over a long period.