Multidrug-resistant tuberculosis.

One of predominant contributors to global mortality is tuberculosis (TB), an infection caused by Mycobacterium tuberculosis (MTB). Inappropriate and ineffectual treatment can lead to the development of drug-resistant TB. One of the most common forms of drug-resistant TB is multidrug-resistant tuberculosis (MDR-TB), caused by mutations in the rpoB and katG genes that lead to resistance to anti-TB drugs, rifampicin (RIF) and isoniazid (INH), respectively. Although culturing remains the gold standard, it is not rapid thereby delaying potential treatment and potentially increasing the incidence of MDR-TB. In contrast, molecular techniques provide a highly sensitive and specific alternative. This review discusses the classification of biomarkers used to detect MDR-TB, some of the commonly used anti-TB drugs, and DNA mutations in MTB that lead to anti-TB resistance. The objective of this review is to increase awareness of the need for rapid and precise detection of MDR-TB cases to decrease morbidity and mortality of this infectious disease worldwide.

Electrochemical profiling of natural furanocoumarins: DNA interaction dynamics of oxypeucedanin and prantschimgin.

Background and purpose: In this study, we present an electrochemical sensor for the detection of oxypeucedanin (Oxyp) and prantschimgin (Pra), two natural furanocoumarin derivatives. The determination of the effects of these molecules on DNA is important to be potential drug candidates. Our research focused on exploring the electrochemical behaviour of these compounds and their interaction with DNA. Experimental approach: The electrochemical properties of Oxyp and Pra were systematically analyzed by evaluating their oxidation currents. Changes in the oxidation currents and peak potentials of guanine bases were monitored before and after interaction in the solution phase and at the electrode surface. Key results: The limit of detection (LOD) and limit of quantitation (LOQ) for Oxyp were determined to be 1.3 and 4.3 µg/mL, respectively. For Pra, the LOD and LOQ were found to be 20 and 68 µg/mL, respectively. Stability studies demonstrated that the Oxyp solution retained its oxidation capacity for over a month, whereas the Pra solution retained its oxidation capacity for nearly 120 min. Our findings suggest that Oxyp interacts with dsDNA, potentially through electrostatic interactions, showing promise as a potential drug candidate targeting DNA. On the other hand, the interaction of Pra with dsDNA requires further exploration to fully understand its mode of action. Conclusion: The electrochemical sensor developed in this study provides a reliable and efficient method for detecting and analysing the interaction of these natural compounds with dsDNA. Our research contributes to advancing the understanding of the interaction between natural furanocoumarins and dsDNA, laying the groundwork for the design and development of novel and effective DNA-targeted drugs.

Expression of scFv-anti-CHIKV-E2 in Escherichia coli with chaperones Co-expression, and its functional assay by electrochemical immunosensor.

Single Chain Variable Fragment (scFv), a small fragment of antibody can be used to substitute the monoclonal antibody for diagnostic purposes. Production of scFv in Escherichia coli host has been a challenge due to the potential miss-folding and formation of inclusion bodies. This study aimed to express anti-CHIKV E2 scFv which previously designed specifically for Asian strains by co-expression of three chaperones that play a role in increasing protein solubility; GroEL, GroES, and Trigger Factor. The scFv and chaperones were expressed in Origami B E. coli host under the control of the T7 promoter, and purified using a Ni-NTA column. Functional assay of anti-CHIKV-E2 scFv was examined by electrochemical immunosensor using gold modified Screen Printed Carbon Electrode (SPCE), and characterized by differential pulses voltammetry (DPV) using K3[Fe(CN)6] redox system and scanning microscope electron (SEM). The experimental condition was optimized using the Box-Behnken design. The results showed that co-expression of chaperone increased the soluble scFv yield from 54.405 µg/mL to 220.097 µg/mL (~5×). Furthermore, scFv can be used to detect CHIKV-E2 in immunosensor electrochemistry with a detection limit of 0.74048 ng/mL and a quantification limit of 2,24388 ng/mL. Thus, the scFv-anti-CHIKV-E2 can be applied as a bioreceptor in another immunoassay method.

Green Synthesis of Ceria Nanoparticles from Cassava Tubers for Electrochemical Aptasensor Detection of SARS-CoV-2 on a Screen-Printed Carbon Electrode.

Green synthesis approaches for making nanosized ceria using starch from cassava as template molecules to control the particle size are reported. The results of the green synthesis of ceria with an optimum calcination temperature of 800 °C shows a size distribution of each particle of less than 30 nm with an average size of 9.68 nm, while the ratio of Ce3+ to Ce4+ was 25.6%. The green-synthesized nanoceria are applied to increase the sensitivity and attach biomolecules to the electrode surface of the electrochemical aptasensor system for coronavirus disease (COVID-19). The response of the aptasensor to the receptor binding domain of the virus was determined with the potassium ferricyanide redox system. The screen-printed carbon electrode that has been modified with green-synthesized nanoceria shows 1.43 times higher conductivity than the bare electrode, while those modified with commercial ceria increase only 1.18 times. Using an optimized parameter for preparing the aptasensors, the detection and quantification limits were 1.94 and 5.87 ng·mL-1, and the accuracy and precision values were 98.5 and 89.1%. These results show that green-synthesized ceria could be a promising approach for fabricating an electrochemical aptasensor.

Hydroxyapatite-Gold Modified Screen-Printed Carbon Electrode for Selective SARS-CoV-2 Antibody Immunosensor.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or coronavirus disease 2019 (COVID-19), is still spreading worldwide; therefore, the need for rapid and accurate detection methods remains relevant to maintain the spread of this infectious disease. Electrochemical immunosensors are an alternative method for the rapid detection of the SARS-CoV-2 virus. Herein, we report the development of a screen-printed carbon electrode immunosensor using a hydroxyapatite-gold nanocomposite (SPCE/HA-Au) directly spray-coated with the immobilization receptor binding domain (RBD) Spike to increase the conductivity and surface electrode area. The HA-Au composite synthesis was optimized using the Box-Behnken method, and the resulting composite was characterized by UV-vis spectrophotometry, TEM-EDX, and XRD analysis. The specific interaction of RBD Spike with immunoglobulin G (IgG) antibodies was evaluated by differential pulse voltammetry and electrochemical impedance spectroscopy methods in a [Fe(CN)6]4-/3- solution redox system. The IgG was detected with a detection limit of 0.0561 pg mL-1, and the immunosensor had selectivity and stability of 103-122% and was stable until week 7 with the influence of storage conditions. Also, the immunosensor was tested using real samples from human serum, where the results were confirmed using the chemiluminescent microparticle immunoassay (CMIA) method and showed satisfactory results. Therefore, the developed electrochemical immunosensor can rapidly and accurately detect SARS-CoV-2 antibodies.

An Electrochemical Sensor of Theophylline on a Boron-Doped Diamond Electrode Modified with Nickel Nanoparticles.

Theophylline is a drug with a narrow therapeutic range. Electrochemical sensors are a potentially effective method for detecting theophylline concentration to prevent toxicity. In this work, a simple modification of a boron-doped diamond electrode using nickel nanoparticles was successfully performed for a theophylline electrochemical sensor. The modified electrode was characterized using a scanning electron microscope and X-ray photoelectron spectroscopy. Square wave voltammetry and cyclic voltammetry methods were used to study the electrochemical behavior of theophylline. The modified nickel nanoparticles on the boron-doped diamond electrode exhibited an electrochemically active surface area of 0.0081 cm2, which is larger than the unmodified boron-doped diamond's area of 0.0011 cm2. This modified electrode demonstrated a low limit of detection of 2.79 µM within the linear concentration range from 30 to 100 µM. Moreover, the modified boron-doped diamond electrode also showed selective properties against D-glucose, ammonium sulfate, and urea. In the real sample analysis using artificial urine, the boron-doped diamond electrode with nickel nanoparticle modifications achieved a %recovery of 105.10%, with a good precision of less than 5%. The results of this work indicate that the developed method using nickel nanoparticles on a boron-doped diamond electrode is promising for the determination of theophylline.

Electrochemical investigation of hydroxyapatite-lanthanum strontium cobalt ferrite composites (HA-LSCF) for SARS-CoV-2 aptasensors.

The hydroxyapatite-lanthanum strontium cobalt ferrite (HA-LSCF) composite showed a good response on a screen-printed carbon electrode (SPCE) electrochemical aptasensor to detect SARS-CoV-2. SPCE/HA-LSCF with a thiolated aptamer has a strong affinity for the SARS-CoV-2 spike RBD protein. This occurs due to the binding of -SH to the HA-positive region. In the presence of LSCF, which is conductive, an increase in electron transfer from the redox system [Fe(CN)6]3-/4- occurs. The interaction of the aptamer with the RBD protein can be observed based on the decrease in the electron transfer process. As a result, the developed biosensor is highly sensitive to the SARS-CoV-2 spike RBD protein with a linear range of 0.125 to 2.0 ng mL-1, a detection limit of 0.012 ng mL-1, and a quantification limit of 0.040 ng mL-1. The analytical application of the aptasensor demonstrates its feasibility in the analysis of saliva or swab samples.

The optimization of electrochemical immunosensors to detect epithelial sodium channel as a biomarker of hypertension.

The epithelial sodium channel (ENaC) is a transmembrane protein that regulates the balance of sodium salt levels in the body through its expression in various tissues. The increase in sodium salt in the body is related to the expression of ENaC, thereby increasing blood pressure. Therefore, overexpression of the ENaC protein can be used as a biomarker for hypertension. The detection of ENaC protein using anti-ENaC in the biosensor system has been optimized with the Box-Behnken experimental design. The steps carried out in this research are screen-printed carbon electrode modification with gold nanoparticles, then anti-ENaC was immobilized using cysteamine and glutaraldehyde. Optimum conditions of the experiment, such as anti-ENaC concentration, glutaraldehyde incubation time, and anti-ENaC incubation time, were optimized using the Box-Behnken experimental design to determine the factors that influence the increase in immunosensor current response and the optimum conditions obtained were then applied to variations in ENaC protein concentrations. The optimum experimental conditions for anti-ENaC concentration were 2.5 µg/mL, the glutaraldehyde incubation time was 30 minutes, and the anti-ENaC incubation time was 90 minutes. The developed electrochemical immunosensor has a detection limit of 0.0372 ng/mL and a quantification limit of 0.124 ng/mL for the ENaC protein concentration range of 0.09375 to 1.0 ng/mL. Thus, the immunosensor generated from this study can be used to measure the concentration of normal urine samples and those of patients with hypertension.

The Optimization of a Label-Free Electrochemical DNA Biosensor for Detection of Sus scrofa mtDNA as Food Adulterations.

Fast, sensitive, and easy-to-use methods for detecting DNA related to food adulteration, health, religious, and commercial purposes are evolving. In this research, a label-free electrochemical DNA biosensor method was developed for the detection of pork in processed meat samples. Gold electrodeposited screen-printed carbon electrodes (SPCEs) were used and characterized using SEM and cyclic voltammetry. A biotinylated probe DNA sequence of the Cyt b S. scrofa gene mtDNA used as a sensing element containing guanine substituted by inosine bases. The detection of probe-target DNA hybridization on the streptavidin-modified gold SPCE surface was carried out by the peak guanine oxidation of the target using differential pulse voltammetry (DPV). The optimum experimental conditions of data processing using the Box-Behnken design were obtained after 90 min of streptavidin incubation time, at the DNA probe concentration of 1.0 µg/mL, and after 5 min of probe-target DNA hybridization. The detection limit was 0.135 µg/mL, with a linearity range of 0.5-1.5 µg/mL. The resulting current response indicated that this detection method was selective against 5% pork DNA in a mixture of meat samples. This electrochemical biosensor method can be developed into a portable point-of-care detection method for the presence of pork or food adulterations.

DNA-based detection of pork content in food.

Determination of halal food is essential in ensuring the tranquillity of consumers, especially Muslims. Halal products mean they are free from prohibited ingredients according to Islamic law. One ingredient that is prohibited is food products containing pork and its derivatives. An accurate verification method with a fast result is necessary to meet this requirement for halal food. DNA quantification of pork is now believed to be able to make accurate and quick decisions, as DNA acts as a reservoir or biological characterization of all living things, including pigs, according to specific characteristics of molecular and connection settings. Various DNA-based methods developed include PCR, biosensor and CRISPR methods. This review discussed various DNA-based Keywords: biosensor, CRISPR, detection, DNA, pork, PCR methods, including PCR, biosensor and CRISPR, to detect pork content in food. Among these methods, CRISPR is considered the easiest, fastest and most accurate. Therefore, it is important to develop this method further in the future. In this article, we provide a short review on DNA-based methods for detection of pork content in food products.

UnpadStat Design: Portable Potentiostat for Electrochemical Sensing Measurements Using Screen Printed Carbon Electrode.

In this research a portable potentiostat was built for electrochemical sensing measurements with three electrodes, specifically SPCEs. The circuit uses a microcontroller as the main controller to manage all activities, starting from adjusting the input voltage for the SPCEs, setting measurement parameters, measuring the resulting current, displaying graphics on the touch screen, sending data to the computer via the USB port, and connecting to the SD card. Measurements and errors with cyclic voltammetry techniques have been compared with commercial potentiostats. The measurement results on a dummy circuit and commercial SPCEs have an accuracy of more than 90% compared to commercial potentiostats. In addition, measurement data can also be saved to an SD card in .CSV format for further purposes.

Smart Graphene-Based Electrochemical Nanobiosensor for Clinical Diagnosis: Review.

The technological improvement in the field of physics, chemistry, electronics, nanotechnology, biology, and molecular biology has contributed to the development of various electrochemical biosensors with a broad range of applications in healthcare settings, food control and monitoring, and environmental monitoring. In the past, conventional biosensors that have employed bioreceptors, such as enzymes, antibodies, Nucleic Acid (NA), etc., and used different transduction methods such as optical, thermal, electrochemical, electrical and magnetic detection, have been developed. Yet, with all the progresses made so far, these biosensors are clouded with many challenges, such as interference with undesirable compound, low sensitivity, specificity, selectivity, and longer processing time. In order to address these challenges, there is high need for developing novel, fast, highly sensitive biosensors with high accuracy and specificity. Scientists explore these gaps by incorporating nanoparticles (NPs) and nanocomposites (NCs) to enhance the desired properties. Graphene nanostructures have emerged as one of the ideal materials for biosensing technology due to their excellent dispersity, ease of functionalization, physiochemical properties, optical properties, good electrical conductivity, etc. The Integration of the Internet of Medical Things (IoMT) in the development of biosensors has the potential to improve diagnosis and treatment of diseases through early diagnosis and on time monitoring. The outcome of this comprehensive review will be useful to understand the significant role of graphene-based electrochemical biosensor integrated with Artificial Intelligence AI and IoMT for clinical diagnostics. The review is further extended to cover open research issues and future aspects of biosensing technology for diagnosis and management of clinical diseases and performance evaluation based on Linear Range (LR) and Limit of Detection (LOD) within the ranges of Micromolar µM (10-6), Nanomolar nM (10-9), Picomolar pM (10-12), femtomolar fM (10-15), and attomolar aM (10-18).

CeO2@NH2 functionalized electrodes for the rapid detection of SARS-CoV-2 spike receptor binding domain.

A detection method based on an electrochemical aptasensor has been developed as an alternative fast, portable, simple, inexpensive, and high-accuracy detection method for detecting the SARS-CoV-2 Spike Receptor Binding Domain (spike RBD). The CeO2@NH2 functionalized Screen Printed Carbon Electrode (SPCE) was used to immobilize an aminated aptamer of spike RBD protein via glutaraldehyde as a linker. The aptamer's interaction with the SARS-CoV-2 Spike RBD was measured via the [Fe(CN)6]4-/3- redox system signal. Experimental conditions were optimized using a Box-Behnken experimental design and showed that the optimal conditions of the SARS-CoV-2 aptasensor were 1.5 ng mL-1 of aptamer, immobilization of aptamer for 60 minutes, and Spike RBD incubation for 10 minutes. The developed aptasensor was able to detect the standard SARS-CoV-2 Spike RBD with a detection limit of 0.017 ng mL-1 in the range of 0.001-100 ng mL-1. This aptasensor was used to detect salivary and oropharyngeal swab samples of normal individuals with the addition of Spike RBD, and the recoveries were 92.96% and 96.52%, respectively. The testing on nasopharyngeal swab samples of COVID-19 patients showed that the aptasensor results were comparable with the qRT-PCR results. Thus, the developed aptasensor has the potential to be applied as a SARS-CoV-2 rapid test method for clinical samples.

An overview of techniques and strategies for isolation of flavonoids from the genus Erythrina.

Plants in the genus Erythrina is a potential source of chemical constituents, one of which is flavonoids, which have diverse bioactivities. To date, literature on the flavonoids from the genus Erythrina has only highlighted the phytochemical aspects, so this review article will discuss isolation techniques and strategies for the first time. More than 420 flavonoids have been reported in the Erythrina genus, which are grouped into 17 categories. These flavonoid compounds were obtained through isolation techniques and strategies using polar, semi-polar, and non-polar solvents. Various chromatographic techniques have been developed to isolate flavonoids using column flash chromatography, quick column chromatography, centrifugally accelerated thin-layer chromatography, radial chromatography, medium-pressure column chromatography, semi-preparative high-performance liquid chromatography, and preparative high-performance liquid chromatography. Chromatographic processes for isolating flavonoids can be optimized using multivariate statistical applications such as response surface methodology with central composite design, Box-Behnken design, Doehlert design, and mixture design.

Low-interference norepinephrine signal on dopamine detection using nafion-coated boron doped diamond electrodes.

The detection of dopamine in the presence of norepinephrine using nafion-coated boron doped diamond (Nafion-BDD) electrodes was presented. An increase current signal for dopamine could be observed at around 0.75 V using Nafion-BDD, while a change in the current signal of norepinephrine that appears at similar potential was not observed. This might be due to electronegativity of the norepinephrine that is not positive enough to be attracted towards the nafion membrane, albeit neutral enough to pass through the membrane and undergo electrochemical oxidation. An optimization process including accumulation time of dopamine inside the nafion layer, solution of the pH, and nafion thickness was conducted to exploit the difference electrochemical behavior between those two catecholamines at the Nafion-BDD. Using an accumulation time of 300 s, solution pH of 7, and nafion thickness of 1.1 µm, dopamine's LOD was found to be 0.966 µM. Low-interference signal of norepinephrine to the dopamine could be observed with an excellent %recovery of dopamine in 5% range when the concentration of norepinephrine was 10 times lower compared to dopamine concentration.

Development of a Single-Chain Variable Fragment of CR3022 for a Plasmonic-Based Biosensor Targeting the SARS-CoV-2 Spike Protein.

Two years after SARS-CoV-2 caused the first case of COVID-19, we are now in the "new normal" period, where people's activity has bounced back, followed by the easing of travel policy restrictions. The lesson learned is that the wide availability of accurate and rapid testing procedures is crucial to overcome possible outbreaks in the future. Therefore, many laboratories worldwide have been racing to develop a new point-of-care diagnostic test. To aid continuous innovation, we developed a plasmonic-based biosensor designed explicitly for portable Surface Plasmon Resonance (SPR). In this study, we designed a single chain variable fragment (scFv) from the CR3022 antibody with a particular linker that inserted a cysteine residue at the second position. It caused the linker to have a strong affinity to the gold surface through thiol-coupling and possibly become a ready-to-use bioreceptor toward a portable SPR gold chip without purification steps. The theoretical affinity of this scFv on spike protein was -64.7 kcal/mol, computed using the Molecular Mechanics Generalized Born Surface Area (MM/GBSA) method from the 100 ns molecular dynamics trajectory. Furthermore, the scFv was produced in Escherichia coli BL21 (DE3) as a soluble protein. The binding activity toward Spike Receptor Binding Domain (RBD) SARS-CoV-2 was confirmed with a spot-test, and the experimental binding free energy of -10.82 kcal/mol was determined using portable SPR spectroscopy. We hope this study will be useful in designing specific and low-cost bioreceptors, particularly early in an outbreak when the information on antibody capture is still limited.

Facile and controllable synthesis of monodisperse gold nanoparticle bipyramid for electrochemical dopamine sensor.

We demonstrated potential features of gold nanoparticle bipyramid (AuNB) for an electrochemical biosensor. The facile synthesis method and controllable shape and size of the AuNB are achieved through the optimization of cetyltrimethylammonium chloride (CTAC) surfactant over citric acid (CA) ratio determining the control of typically spherical Au seed size and its transition into a penta-twinned crystal structure. We observe that the optimized ratio of CTAC and CA facilitates flocculation control in which Au seeds with size as tiny as ∼14.8 nm could be attained and finally transformed into AuNB structures with an average length of ∼55 nm with high reproducibility. To improve the electrochemical sensing performance of a screen-printed carbon electrode, surface modification with AuNB via distinctive linking procedures effectively enhanced the electroactive surface area by 40%. Carried out for the detection of dopamine, a neurotransmitter frequently linked to the risk of Parkinson's, Alzheimer's, and Huntington's diseases, the AuNB decorated-carbon electrode shows outstanding electrocatalytic activity that improves sensing performance, including high sensitivity, low detection limit, wide dynamic range, high selectivity against different analytes, such as ascorbic acid, uric acid and urea, and excellent reproducibility.

Detection of Tropical Diseases Caused by Mosquitoes Using CRISPR-Based Biosensors.

Tropical diseases (TDs) are among the leading cause of mortality and fatality globally. The emergence and reemergence of TDs continue to challenge healthcare system. Several tropical diseases such as yellow fever, tuberculosis, cholera, Ebola, HIV, rotavirus, dengue, and malaria outbreaks have led to endemics and epidemics around the world, resulting in millions of deaths. The increase in climate change, migration and urbanization, overcrowding, and other factors continue to increase the spread of TDs. More cases of TDs are recorded as a result of substandard health care systems and lack of access to clean water and food. Early diagnosis of these diseases is crucial for treatment and control. Despite the advancement and development of numerous diagnosis assays, the healthcare system is still hindered by many challenges which include low sensitivity, specificity, the need of trained pathologists, the use of chemicals and a lack of point of care (POC) diagnostic. In order to address these issues, scientists have adopted the use of CRISPR/Cas systems which are gene editing technologies that mimic bacterial immune pathways. Recent advances in CRISPR-based biotechnology have significantly expanded the development of biomolecular sensors for diagnosing diseases and understanding cellular signaling pathways. The CRISPR/Cas strategy plays an excellent role in the field of biosensors. The latest developments are evolving with the specific use of CRISPR, which aims for a fast and accurate sensor system. Thus, the aim of this review is to provide concise knowledge on TDs associated with mosquitoes in terms of pathology and epidemiology as well as background knowledge on CRISPR in prokaryotes and eukaryotes. Moreover, the study overviews the application of the CRISPR/Cas system for detection of TDs associated with mosquitoes.

A review post-vaccination SARS-CoV-2 serological test: Method and antibody titer response.

The development of the Coronavirus disease 2019 (COVID-19) vaccine is one of the most important efforts in controlling the pandemic. Serological tests are used to identify highly reactive human donors for convalescent plasma therapy, measuring vaccine efficacy and durability. This review article presents a review of serology tests and how antibody titers in response to vaccines have been developed. Some of the serological test methods discussed are Plaque Reduction Neutralization Test (PRNT), Enzyme-Linked Immunosorbent Assay (ELISA), Lateral flow immunoassay (LFIA), chemiluminescent immunoassay (CLIA), and Chemiluminescent Micro-particle Immunoassay (CMIA). This review can provide an understanding of the application of the body's immune response to vaccines to get some new strategies for vaccines.

Enhanced adsorption capacity of activated carbon over thermal oxidation treatment for methylene blue removal: kinetics, equilibrium, thermodynamic, and reusability studies.

Activated carbon (AC) is an effective and inexpensive adsorbent material for dye removal, but it cannot always be used repeatedly. Furthermore, the adsorbed dyes with toxicity usually remain on its surface. In this study, a thermal air oxidation process was used to modify the surface of AC and decompose adsorbed methylene blue (MB). The behavior of this process on spent AC was investigated using TGA-DTA, while the degradation of MB before and after the regeneration process was analyzed using a carbon, hydrogen, nitrogen, sulfur (CHNS) analyzer. It was discovered that thermal air oxidation could promote the formation of oxygenated functional groups on AC produced from steam-activated carbon coconut shell (SACCS), which when treated at 350 °C (denoted as SACCS-350), demonstrated an adsorption capacity 2.8 times higher than the non-air-oxidized AC (SACCS). The key parameters for the MB adsorption of SACCS and SACCS-350, such as kinetics, equilibrium, and thermodynamics, were compared. Moreover, the SACCS-350 could be reused at least 3 times for the adsorption of MB. Based on these results, thermal air oxidation treatment could successfully improve the adsorption performance of AC and regenerate spent AC through a reasonable and environmentally friendly process compared to other regeneration methods.