When it comes to diagnostics for various microorganisms, biosensors offer great advantages over conventional analytical techniques. Specifically, they can provide multiple capabilities such as user-friendly operation, real-time analysis, rapid response, high sensitivity and specificity, portability, label-free detection, and cost-effectiveness. As a result, this diagnostic approach possesses suitable features to develop point‐of‐care (POC) diagnostics and monitoring technologies. In this study, for the first time, an optical biosensor chip was developed and analysed using a localised surface plasmon resonance (LSPR) optical biosensing technique to monitor biomolecular interactions between mycolic acid TB antigen and anti-mycobacterium tuberculosis antibody. Mycolic acid was successfully immobilised on a gold-coated biosensor chip and allowed to react with an anti-mycobacterium tuberculosis antibody. To enhance the detection signal from biomolecular binding events, AuNPs were used and successfully bioconjugated with goat anti-rabbit IgG H&L secondary antibody and characterised using ultraviolet-visible (UV-vis) spectroscopy and subsequently introduced on the biosensing layer. Scanning electron microscopy (SEM) and energy-dispersive x-ray (EDX) spectroscopy were used to characterise the biosensing surface. The optimised biosensor chip was analysed using a custom-built biosensing transmission spectroscopy setup to perform LSPR biosensing. From our findings, it was realised that mycolic acid was successfully immobilised on the biosensing surface and made it possible to capture anti-mycobacterium tuberculosis antibodies. The LSPR optical biosensing technique was indeed successful in the detection of anti-mycobacterium tuberculosis antibodies.
Manipulation of biological cells using optical trapping is a non-invasive approach in which individual living cells are examined without causing any damage because there is no direct mechanical contact with cells. Optical trapping uses a tightly focused laser beam emitted through a high numerical aperture microscope objective lens to hold microscopic particles. When using this technique, there is minimal chances of exposing cells to contamination and optically handled cells can still be utilised in downstream sterile experiments whenever necessary. In this study, optical trapping is used to trap HIV infected cells, which are then analysed by Raman spectroscopy. Raman spectroscopy as an analytical technique provides specific chemical/molecular details about a sample based on the fundamental vibrational modes of the chemicals. By combining these two light-based technologies, HIV infected TZM-bl cells were distinguished from the uninfected cells as they exhibited different molecular fingerprints. The acquired results both confirm and provide more detail to the findings of the previous study where transmission spectroscopy was used to differentiate between HIV infected and uninfected cells. This current study shows how the two cell populations differ according to the chemical/molecular composition and distribution. These results present valuable information that would be essential in the development of a label-free HIV point of care diagnostic device.
Recently, various nanomaterials have been used to develop nanotechnology‐based rapid diagnostic tests. Due to their unique optical properties, gold nanoparticles (AuNPs) have been employed to design and develop modern biosensors for the rapid and real-time detection of various diseases or pathogen‐specific biomolecules/markers, such as DNA, RNA, proteins, and whole cells. Optical biosensors offer great advantages over conventional analytical techniques. Specifically, they can provide multiple capabilities such as user-friendly operation, real-time analysis, rapid response, high sensitivity and specificity, portability, label-free detection and cost-effectiveness. As a result, this diagnostic approach possesses suitable features to develop point‐of‐care (POC) diagnostics and monitoring technologies. This study implemented the use of surface plasmon resonance (SPR) biosensing to monitor biomolecular interaction between biorecognition element covalently immobilized on a gold-coated glass substrate and an analyte. A custom-built Kretschmann configuration SPR optical biosensing setup was used to measure angle shift to monitor the biomolecular interaction events on the biosensing layer. To amplify the differences in SPR biosensing due to biomolecular binding events, AuNPs were used and successfully conjugated to the anti-TB antibodies and confirmed using ultraviolet–visible (UV-vis) spectroscopy. Mycolic acids were successfully immobilized on gold-coated substrates and were able to bind to the anti-TB antibodies that were introduced on the substrates, therefore enabling the detection of the captured anti-TB antibodies. As a result, mycolic acids have been realized to be efficient biomarkers to specifically react with anti-TB antibodies and produce a detectable signal for the purpose of TB diagnosis.
In recent years, conjugated nanoparticles have gained significant applications in diagnostics, particularly gold nanoparticles (AuNPs). When functionalized with antibodies, AuNPs can selectively interact with cells and biomolecules. The conjugation of biomolecules to AuNPs has been achieved using a variety of techniques, one such approach is the covalent coupling method used in the current study. Generally, in diagnostics, the conjugation of different moieties such as antibodies to the AuNPs widens their applications and provides them with new or enhanced properties. Due to their high specificity and diversity, antibodies are widely used to provide specificity and bioactivity to AuNPs, particularly for biosensor applications. Localized surface plasmon resonance (LSPR) has emerged as a leader among label-free biosensing techniques because it offers sensitive, robust, and rapid detection of biological analytes. Biomolecular adsorptions on AuNPs surface increases the dielectric constant and change the intensities and the wavelengths of the LSPR band associated with AuNPs. As a result, the adsorptions of biomolecules onto surfaces of this AuNPs can be monitored by measuring the absorption spectra of the AuNPs. In this study, TB antibodies were covalently conjugated to AuNPs and used to detect mycolic acid TB antigens at various concentrations. Characterization of the AuNPs was done using transmission electron microscopy (TEM) while the biomolecular interaction between TB antibodies and the antigen was measured using LSPR. From our findings, it was realised that the use of antibodyconjugated AuNPs enhanced the detection of the analyte even at low concentrations of the analyte.
Surface plasmon resonance (SPR) biosensors are optical materials that measure changes in the refractive index as they monitor non-covalent molecular interactions in real time. These utilise a label free analytical approach, which does not require dyes to produce a visible signal. In this study SPR was assessed for the detection of DNA hybridization between complementary DNA sequences within the pol gene of the human immunodeficiency virus (HIV) genome. HIV mutates rapidly due to its error prone reverse transcriptase enzyme. Some of these mutations make the virus to be resistant to antiretroviral drugs used to treat HIV infected individuals, rendering the drugs ineffective. In order to assess whether an infected individual expresses any drug resistant mutations, different bio-assays must be performed. However, these tests are expensive and require sophisticated equipment, which might be unavailable in resource limited settings. In a quest to simplify these tests so that they can be used in resource limited settings and reduce costs associated with HIV drug resistance testing, SPR capabilities were explored in this study. This was achieved by amplifying a 174 bp region of the HIV-1 pol gene using polymerase chain reaction (PCR). The detection was based on the hybridization between the PCR amplified DNA sequence and a biotinylated oligonucleotide probe immobilized onto an SPR sensor chip made of a gold coated slide. The acquired results indicated that the SPR-sensor-chip used was able to recognize changes in different wells and thereby able to differentiate between a sample with DNA hybridization and the one without. Based on these findings, this approach has potential to detect HIV drug resistance mutations with high efficiency in less time, at lower cost.
Polypeptide gelatine has been used extensively in microbiology to enhance cellular adhesion and growth. Likewise, fabrication of biochemical sensors using a variety of organic material and nanomaterials is a growing research area particularly in experiments involving single molecular screening. Both fields of study exploit the various interactions that occur at molecular level such as charge-charge binding, hydrogen bonding and van Der Waals forces. In this work, a thin film gelatine based biosensor, containing amino acids such as glycine, proline and hydroxy-proline was synthesized on glass slides using the self-assembly method. Further -adaption involved coating gold nanoparticles onto the substrate to enhance chemical binding and improve signal intensity and sensitivity. Pharmaceutical drugs aspirin and paracetamol were used as analytes to explore the qualitative and quantitative capabilities of the sensor in molecular screening through surface enhanced Raman spectroscopy (SERS). The results showed a distinguishable qualitative difference between the Raman spectra of gelatine-drug (Gel-D) and gelatine-gold-drug (Gel-Au-D) fabricated sensors. Similarly in both Gel-D and Gel-Au-D, the peak areas of the functional groups found in both aspirin and paracetamol increased with drug concentration, yielding satisfactory calibration curves. The gelatine based biosensor thus holds potential as an in vitro sensing platform for screening of pharmaceutical drugs.
Surface Plasmon Resonance (SPR) which is widely used to study interactions between different types of biomolecules, has emerged as a technique of choice for rapid and quantitative analyses. However, there are still some challenges on the use of the classical SPR optical configuration. The prism-based configuration setup requires precise alignment of light onto the sample surface and the oblique reflection angle plane yield optical aberration. In this work we have built, characterized and optimized a simple collinear transmission geometry plasmonic system for the detection of HIV-1. Here, a continuous wave laser at 785 nm with power output of 300 mW was used as light source and a 40X objective lens coupled to a CCD camera was used to collect and detect the transmitted intensity change. Furthermore, a white light source was used to study the wavelength dependency of the sample. We present our findings which may be useful to develop biomedical devices for point-of-care diagnostics and healthcare applications.
Biological macromolecules such as antibodies, enzymes, proteins and aptamers have good molecular recognition ability which makes them good candidates for biosensing applications. In this study, glass substrates were treated with silane in order to immobilize HIV gp41 antibodies on their surfaces. The HIV pseudovirus was added to the treated substrates followed by addition of antibodies conjugated to nanoparticles. The surfaces were characterised by using water contact angle, atomic force microscopy (AFM) and Raman spectroscopy. Our preliminary data displayed that the antibodies were indeed immobilized on the glass substrates which made it possible for capturing the intact HIV pseudovirus. Further, Raman spectroscopy revealed the presence of disulphide bonds indicating successful conjugation of the HIV gp41 antibodies to the HIV pseudovirus.
A lot of individuals residing in resource limited settings where timely access to medical care is a challenge and healthcare infrastructure is usually poor have no access to laboratory facilities. Disease diagnosis in such sites is dependent on the presence of point-of-care (POC) devices. These POC diagnostics play a key role in ensuring rapid patient care because they are simple to use, inexpensive, portable, instrument independent and do not require a trained technician to operate. In this study, we used a smartphone camera as a spectrometer for measurement of rhodamine at different concentrations. Rhodamine was used as the analyte of choice for proof of concept purposes. The smartphone platform was able to detect the absorption within the visible spectral range from 400 to 700 nm. The results obtained showed that the performance of the smartphone based platform correlates with the conventional microplate reader. From this study, we therefore envision an inexpensive and portable smartphone based devise with connectivity to the internet for POC diagnostics in resource limited settings.
Accurate sorting of specific particles in a mixed population is a desirable capability in the field of biomedical sciences. This enables researchers to purify samples by selecting only the particles of interest. Optical sorting is achieved by using a Bessel beam, which is a non-diffracting, propagation invariant light pattern consisting of concentric rings around a bright central core. This type of beam profile has the ability to employ optical forces in manipulating matter in a sterile environment without physical interaction. The concentric rings enable the simultaneous manipulation of particles of various characteristics in multiple planes due to the different power intensity distributions. Sorting with Bessel beam is an attractive approach using small sample volumes (microliter ranges), which becomes beneficial when working with rare particles of interest and in small samples. In this study a home built Bessel beam optical sorting setup was used to sort polystyrene and silica microspheres of different sizes and refractive indices. Our preliminary results showed that the polystyrene microspheres travelled quicker than the silica type of spheres with the same size due to the high refractive indices. These findings indicate the potential application of sorting different cells with varying refractive indices such as differentiating HIV infected cells from uninfected cells.
Raman spectroscopy is commonly used for sample characterization in biology because vibrational information is very specific to the chemical bonds in molecules. This makes it an attractive approach for identification of biological materials such as toxins, viruses or even intact bacterial cells. In addition, Raman spectroscopy has a unique capability of providing label-free intrinsic chemical information, such as molecular bonds in living biological samples at tissue, cellular or subcellular resolution. However, Raman signals are weak and acquiring a spectrum with good signal to noise ratio requires long acquisition time. To overcome this disadvantage of low signal intensities from most biomolecules, enhancement effects are utilized. In this study, a home built Raman spectroscopy optical system combined with a gold thin film deposition was used to detect the HIV gp41 antibody. The Raman system makes use of 785 nm diode laser as excitation source and an Andor CCD camera as detection system. In addition, we report on Raman results obtained with HIV gp41 antiboby using a gold thin film deposition substrate. We could observe significant enhancement of Raman signal from the gold thin film layer deposition. These findings indicate the potential application of Raman spectroscopy in rapid biosensing detection.
An optical biosensor is a compact analytical device formed by a bio-recognition sensing element integrated to an optical transducer system which translates a signal into a readable outcome that is measured by the detector. The target analyte interacts with an immobile bio-recognition element giving rise to a signal proportionate to the concentration of a measured analyte. Optical biosensors offer great advantages over conventional analytical techniques. Specifically, they can provide multiple capabilities such as user-friendly operation, real-time analysis, rapid response, high sensitivity and specificity, portability, label-free detection and cost-effectiveness. As a result, they possess suitable features critical for point-of-care diagnostics. In this study, a home-build surface plasmon resonance (SPR) optical biosensor device was used to analyse interactions between the bio-recognition sensing element and an analyte on the biosensing layer. The transducer consisted of silica dioxide (SiO2) substrate layer where a thin layer of gold was deposited. Mycolic acid antigens from mycobacterium tuberculosis (bovine strain) were immobilised on the biosensing layer and used as biorecognition sensing elements to capture tuberculosis (TB) antibodies (analyte). From our findings, it was realised that the mycolic acid successfully captured TB antibodies resulting in a detectable signal which paves a way for the development of the point-of-care device.
Surface enhanced Raman spectroscopy (SERS) has evolved to be a powerful analytical tool for investigating molecular properties of various types of samples. Literature has shown SERS capabilities in both qualitative and quantitative analysis of biomolecules like proteins and DNA as well as single molecules like antiretroviral medication. Central to its application is the synthesis and use of sensing platforms that enhance signal intensity, sensitivity and detection limits. The most popular approach to make such platforms is through fabricating thin film substrates using a combination of polymers and nanomaterials. In this work, we use the self-assembly method to synthesize graphene oxide based scaffolds in a layer-by-layer fashion and characterize them using SERS. The results show a clear difference in Raman spectral fingerprint for the different layers during the self-assembly steps. Lastly, the intensity ratio between the D and G bands of the graphene layer were calculated to measure the layer thickness which was found to be 0.65, this was comparable to thin layer scaffolds reported in literature. Future work will involve the use of atomic force microscopy to confirm surface morphology and layer thickness, followed by screening of antiretroviral medication.
Surface Plasmon Resonance (SPR) is a very powerful optical sensing technique that detects bimolecular binding interactions and it has turned out to be a suitable platform for clinical analysis. In biological and chemical sensing applications, SPR is used to monitor molecular binding real-time and it also promotes epitope mapping for determining biomolecular structures such as the interactions of proteins, DNA and viruses. This sensing technique also provides sensitive, label free and real-time monitoring of reactions. In this study we have built, characterized and optimized the SPR system for biosensing applications. Spectroscopy and scanning electron microscopy were used to characterize the surface of the SPR biosensor chip functionalized with antibodies. The home-built SPR system was successful in detecting biological analytes thereby paving a way into designing a label-free point-of-care (POC) diagnostic tool.
Gold thin metal layers have been seen to be the most important signal amplification components in electrochemical and optical sensor applications. In surface plasmon resonance (SPR) applications, gold thin metal film possess electron densities that have the plasmon frequencies in the visible light range. In this study, gold thin film layer coating was deposited onto a glass substrate by using the ebeam physical vapour deposition technique. The structural and morphological investigations of the thin film layer coating were investigated using the X-ray diffraction system (XRD) while the scanning electron microscope (SEM) was used to investigate the morphology of the thin film layer coating. The optical analysis using different thicknesses, showed good absorption and transmission spectra that is required in determining the appropriate layer to be used for surface plasmon resonance. Using the XRD, SEM as well as transmission and absorption spectroscopy, the findings indicated that gold thin film layer deposition using the ebeam evaporation system is perfect for thin film layer coating for surface plasmon resonance applications.
Over the past few years gold nanoparticle (AuNPs) have become extremely interesting because they possess enhanced optical, electrical and chemical properties. AuNPs have the ability to form robust conjugates with biomolecules such as antibodies and can enhance optical signals making them suitable for a variety of diagnostic applications including the surface plasmon resonance (SPR) technique. SPR is a highly sensitive and label free optical technique which is widely used for detecting biological analytes and analysing the interaction between different types of biomolecules. In this study, bioconjugation was achieved by covalently attaching antibodies to AuNPs and gold coated slides were used as SPR sensor chips in Kretschmann configuration. Several UV/VIS excitation spectra were collected before and after AuNPs were conjugated to antibodies. The results showed that the sensitivity of the SPR system significantly increased because of the bioconjugation of antibodies to AuNPs and this is a promising approach for biosensing applications.
Photonic crystals (PhCs) is a unique and flexible class of optical devices that are able to manipulate the electromagnetic fields of light. PhCs is a subwavelength grating structure with a periodic arrangement of a high refractive index layer coated on a low refractive index material and can provide a strong light confinement depending on the size, periodicity and the refractive index. Finite difference time domain (FDTD) method can be used to simulate the electromagnetic properties of light through complex structures such as PhCs, because of the precision of the method in the description of geometry and properties of the material. In this study, FDTD software from Lumerical was used to design and simulate the electromagnetic properties of the PhCs based sensor for biosensing applications. The transmission, reflection and absorption characteristics through the proposed PhCs structure was analysed using a visible wavelength range of 400- 700 nm. The boundary conditions were correctly chosen and consisted of periodic boundary conditions and perfectly matched layers. The results revealed that the transmission and reflectance were dependent on the period of the PhCs and the enhanced electric field was confined in an area allowing for interaction with biological analytes.
The use of light to inactivate microbes as an alternative method to the traditional methods of controlling microorganisms continues to draw the attention of researchers. Traditional methods of sterilization and/or pasteurization using chemicals or thermal treatments have certain limitations such as the creation of resistant bacterial strains. The application of pulsed laser irradiation compromises the physiological function of cells, and the degree of destruction is both dose and strain dependent, ranging from reduced cell growth to a complete loss of cell metabolic activity and finally to physical disintegration. This study aimed at using a range of power densities to investigate inactivation of Escherichia coli and Salmonella enteritidis. A Titanium sapphire pulsed laser at 800 nm wavelength, repetition rate of 76 MHz, pulse duration of 120 fs, output power of 560 mW was used in this study. A fluence range was applied on bacterial cultures in a 16 mm diameter petri with a beam spot area of 2.5 cm2 (after expansion). The laser killing effectiveness was evaluated by comparing colony forming units (CFUs) with and without irradiation on 10-7 dilutions of bacterial cultures. Cytotoxicity was analysed using the lactose dehydrogenase (LDH) assay. The laser killing rate varied with bacteria species or strains and the level of fluence.
In tissue engineering research, stem cells have been used as starting material in the synthesis of mammalian cells for the treatment of various cell based diseases. This is done by manipulating the DNA content of the cells to induce a specific effect such as increased proliferation or developing a new cell type through the process of differentiation. Such controlled gene expression of stem cells is achieved by the method of transfection, where exogenous plasmid deoxyribonucleic acid (pDNA) is inserted into a stem cell using chemical, viral or physical methods. In this research, we used femtosecond (fs) laser pulses from a home-build microscope system to perforate the cellular membrane and allow entry of selected pDNA to alter the behaviour of mouse embryonic stem cells (mESCs). In one set of experiments, we induce fluorescence on mESCs using green fluorescence protein plasmid (pGFP) while in other tests; differentiation of mESCs into endoderm cells is performed using Sox-17 plasmid DNA (pSox-17). Primitive endoderm formation was thereafter confirmed using polymerase chain reactions (PCR) and the Sox-17 primer. Cell viability studies using adenosine triphosphate were also conducted. From the data, it was concluded that the photo-transfection method is biocompatible since it was able to induce fluorescence in mESCs. Secondly, it was confirmed that Sox-17 was photo-transfected successfully using 6 μW laser power, 128 fs pulses and 1kHz pulse repetition rate.
Optical trapping has emerged as an essential tool for manipulating single biological material and performing sophisticated spectroscopy analysis on individual cell. The optical trapping technique has been used to grab and immobilize cells from a tightly focused laser beam emitted through a high numerical aperture objective lens. Coupling optical trapping with other technologies is possible and allows stable sample trapping, while also facilitating molecular, chemical and spectroscopic analysis. For this reason, we are exploring laser trapping combined with laser spectroscopy as a potential non-invasive method of interrogating individual cells with a high degree of specificity in terms of information generated. Thus, for the delivery of as much pathological information as possible, we use a home-build optical trapping and spectroscopy system for real time probing human immunodeficiency virus (HIV-1) infected and uninfected single cells. Briefly, our experimental rig comprises an infrared continuous wave laser at 1064 nm with power output of 1.5 W, a 100X high numerical aperture oil-immersion microscope objective used to capture and immobilise individual cell samples as well as an excitation source. Spectroscopy spectral patterns obtained by the 1064 nm laser beam excitation provide information on HIV-1 infected and uninfected cells. We present these preliminary findings which may be valuable for the development of an HIV-1 point of care detection system.
Human immunodeficiency virus (HIV-1) infection remains a major health problem despite the use of highly active antiretroviral therapy (HAART), which has greatly reduced mortality rates. Due to the unavailability of an effective vaccine or a treatment that would completely eradicate the virus, the quest for new and combination therapies continues. In this study we explored the influence of Low Level Laser Therapy (LLLT) in HIV-1 infected and uninfected cells. Literature reports LLLT as widely used to treat different medical conditions such as diabetic wounds, sports injuries and others. The technique involves exposure of cells or tissue to low levels of red and near infrared laser light. Both HIV infected and uninfected cells were laser irradiated at a wavelength of 640 nm with fluencies ranging from 2 to 10 J/cm2 and cellular responses were assessed 24 hours post laser treatment. In our studies, laser therapy had no inhibitory effects in HIV-1 uninfected cells as was indicated by the cell morphology and proliferation results. However, laser irradiation enhanced cell apoptosis in HIV-1 infected cells as the laser fluencies increased. This led to further studies in which laser irradiation would be conducted in the presence of HAART to determine whether HAART would minimise the detrimental effects of laser irradiation in infected cells.
Embryonic stem cells have great promise in regenerative medicine because of their ability to self-renew and differentiate into various cell types. Delivery of therapeutic genes into cells has already been achieved using of chemical agents and viral vectors with high transfection efficiencies. However, these methods have also been documented as toxic and in the latter case they can cause latent cell infections. In this study we use femtosecond laser pulses to optically deliver genetic material in mouse embryonic stem cells. Femtosecond laser pulses in contrast to the conventional approach, minimises the risk of unwanted side effects because photons are used to create transient pores on the membrane which allow free entry of molecules with no need for delivery agents. Using an Olympus microscope, fluorescence imaging of the samples post irradiation was performed and decreased expression of stage specific embryonic antigen one (SSEA-1) consistent with on-going cellular differentiation was observed. Our results also show that femtosecond laser pulses were effective in delivering SOX 17 plasmid DNA (pSOX17) which resulted in the differentiation of mouse embryonic stem cells into endoderm cells. We thus concluded that laser transfection of stem cells for the purpose of differentiation, holds potential for applications in tissue engineering as a method of generating new cell lines.
The current human immunodeficiency virus (HIV) treatment regime possesses the ability to diminish the viral capacity to unnoticeable levels; however complete eradication of the virus cannot be achieved while latent HIV-1 reservoirs go unchallenged. Therapeutic targeting of HIV therefore requires further investigation and current therapies need modification in order to address HIV eradication. This deflects research towards investigating potential novel antiretroviral drug delivery systems. The use of femtosecond (fs) laser pulses in promoting targeted optical drug delivery of antiretroviral drugs (ARVs) into TZMbl cells revolves around using ultrafast laser pulses that have high peak powers, which precisely disrupt the cell plasma membrane in order to allow immediate transportation and expression of exogenous material into the live mammalian cells. A photo-translocation optical setup was built and validated by characterisation of the accurate parameters such as wavelength (800 nm) and pulse duration (115 fs). Optimisation of drug translocation parameters were done by performing trypan blue translocation studies. Cellular responses were determined via cell viability (Adenosine Triphosphate activity) and cell cytotoxicity (Lactate Dehydrogenase) assays which were done to study the influence of the drugs and laser exposure on the cells. After laser irradiation, high cell viability was observed and low toxicity levels were observed after exposure of the cells to both the ARVs and the laser. Our results confirmed that, with minimal damage and high therapeutic levels of ARVs, the fs laser assisted drug delivery system is efficient with benefits of non-invasive and non-toxic treatment to the cells.
Cellular manipulation by delivery of molecules into cells has been applied extensively in tissue engineering research for medical applications . The different molecular delivery techniques used range from viral and chemical agents to physical and electrical methods. Although successful in most studies, these techniques have inherent difficulties such as toxicity, unwanted genetic mutations and low reproducibility respectively. Literature recognizes pulsed lasers at femtosecond level to be most efficient in photonic interactions with biological material. As of late, laser pulses have been used for drug and DNA delivery into cells via transient optical perforation of the cellular membrane. Thus in this study, we design and construct an optical system coupled to a femtosecond laser for the purpose of phototransfection or insertion of plasmid DNA (pDNA) into cells using lasers. We used fluorescent green protein (pGFP) to transfect mouse embryonic stem cells as our model. Secondly, we applied fluorescence imaging to view the extent of DNA delivery using this method. We also assessed the biocompatibility of our system by performing molecular assays of the cells post irradiation using adenosine triphosphate (ATP) and lactate dehydrogenase (LDH).
Human immunodeficiency virus (HIV-1) infection still remains one amongst the world’s most challenging infections since its discovery. Antiretroviral therapy is the recommended treatment of choice for HIV-1 infection taken by patients orally. The highly active antiretroviral therapy (HAART) prevents the replication of HIV-1 and further destruction of the immune system, therefore enabling the body to fight opportunistic life-threatening infections, cancers, and also arrest HIV infection from advancing to AIDS. The major challenge with HAART is the inability to reach the viral reservoirs where the HIV-1 remains latent and persistent, leading to inability to fully eradicate the virus. This study is aimed at initially designing and assembling a fully functional optical translocation setup to optically deliver antiretroviral drugs into HIV-1 infected cells in a targeted manner using Gaussian beam mode femtosecond laser pulses in-vitro. The main objective of our study is to define the in-vitro drug photo-translocation parameters to allow future design of an efficient drug delivery device with potential in-vivo drug delivery applications. In our experiments, HEK 293T cells were used to produce HIV-1 enveloped pseudovirus (ZM53) to infect TZM-bl cells which were later treated with laser pulses emitted by a titanium sapphire laser (800 nm, 1KHz, 113 fs, ~ 6.5 μW) to create sub-microscopic pores on the cell membrane enabling influx of extracellular media. Following laser treatment, changes in cellular responses were analysed using cell morphology studies, cytotoxicity, and luciferase assay studies. Controls included laser untreated cells incubated with the drug for 72 hours. The data in this study was statistically analysed using the SigmaPlot software version 13.
Photodynamic therapy (PDT) is a minimally invasive therapeutic modality for the treatment of neoplastic and
non-neoplastic diseases. In PDT of cancer, irradiation with light of a specific wavelength leads to activation of
a photosensitizer which results in generation of reactive oxygen species (ROS) which induces cell death.
Many phthalocyanine photosensitizers are hydrophobic and insoluble in water, which limits their therapeutic
efficiency. Consequently, advanced delivery systems and strategies are needed to improve the effectiveness
of these photosensitizers. Nanoparticles have shown promising results in increasing aqueous solubility,
bioavailability, stability and delivery of photosensitizers to their target. This study investigated the
photodynamic activity of zinc monocarboxyphenoxy phthalocyanine (ZnMCPPc) conjugated to gold silver
(AuAg) nanoparticles in melanoma cancer cells. The photodynamic activity of ZnMCPPc conjugated to AuAg
nanoparticles were evaluated using cellular morphology, viability, proliferation and cytotoxicity. Untreated cells
showed no changes in cellular morphology, proliferation and cytotoxicity. However, photoactivated ZnMCPPc
conjugated to AuAg nanoparticles showed changes in cell morphology and a dose dependent decrease in
cellular viability, proliferation and an increase in cell membrane damage. The ZnMCPPc conjugated to AuAg
nanoparticles used in this study was highly effective in inducing cell death of melanoma cancer cells.
Photodynamic therapy (PDT) has emerged as an effective treatment modality for various malignant neoplasia
and diseases. In PDT, the photochemical interaction of photosensitizer (PS), light and molecular oxygen
produces singlet oxygen which can lead to tumour cell apoptosis, necrosis or autophagy. The success of PDT
is limited by the hydrophobic characteristic of the PS which hinders treatment administration and efficiency.
To circumvent this limitation, PS can be incorporated in nanostructured drug delivery systems such as gold
nanoparticles (AuNPs). In this study, we investigated the effectiveness of free zinc monocarboxyphenoxy
phthalocyanine (ZnMCPPc) and ZnMCPPc conjugated to AuNPs. Commercially purchased melanoma cancer
cells cultured as cell monolayers were used in this study. Changes in cellular response were evaluated using
cellular morphology, viability, proliferation and cytotoxicity. Untreated cells showed no changes in cellular
morphology, proliferation and cytotoxicity. However, photoactivated free ZnMCPPc and ZnMCPPc conjugated
to AuNPs showed changes in cellular morphology and a dose dependent decrease in cellular viability and
proliferation as well as an increase in cell membrane. ZnMCPPc conjugated to AuNPs showed an improved
efficiency in PDT as compared to free ZnMCPPc, which might be as a result of the vehicle effect of AuNPs.
Both PSs used in this study were effective in inducing cell death with ZnMCPPc conjugated to AuNPs
showing great potential as an effective PS for PDT.
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