Fungal Biosensors Research Articles

Simple and rapid detection of Alternaria alternata using an excreted redox‐active species

AbstractAirborne fungi pose a serious threat to public health. Alternaria alternata (A. alternata) is a fungus that has been associated with the development of asthma. Detection using redox‐active species excreted from fungi is an effective method for a simple electrochemical fungal biosensor. The achievable electrochemical signal in most fungi, however, is exceptionally low because of the low amount of excreted redox‐active species and their slow excretion rates. Herein, we report that A. alternata excretes an exceptionally large amount of a redox‐active species that can be used for sensitive and selective detection of A. alternata. The excretion rate is enhanced in Tris buffer, and the electrochemical‐chemical redox cycling involving excreted redox‐active species significantly increases the electrochemical signals. Only A. alternata among five common airborne fungi provides large electrochemical signals, which allows selective detection of A. alternata. The calculated detection limit for A. alternata is ~20 spores/mL with an incubation period of 10 min, indicating that the detection method is highly sensitive and rapid. The detection method does not require complicated procedures or harsh pretreatment and is optimal for point‐of‐care testing of A. alternata.

Biosensors and Diagnostics for Fungal Detection.

Early detection is critical to the successful treatment of life-threatening infections caused by fungal pathogens, as late diagnosis of systemic infection almost always equates with a poor prognosis. The field of fungal diagnostics has some tests that are relatively simple, rapid to perform and are potentially suitable at the point of care. However, there are also more complex high-technology methodologies that offer new opportunities regarding the scale and precision of fungal diagnosis, but may be more limited in their portability and affordability. Future developments in this field are increasingly incorporating new technologies provided by the use of new format biosensors. This overview provides a critical review of current fungal diagnostics and the development of new biophysical technologies that are being applied for selective new sensitive fungal biosensors to augment traditional diagnostic methodologies.

A robust high-throughput fungal biosensor assay for the detection of estrogen activity

Estrogenic active compounds are present in a variety of sources and may alter biological functions in vertebrates. Therefore, it is crucial to develop innovative analytical systems that allow us to screen a broad spectrum of matrices and deliver fast and reliable results. We present the adaptation and validation of a fungal biosensor for the detection of estrogen activity in cow derived samples and tested the clinical applicability for pregnancy diagnosis in 140 mares and 120 cows. As biosensor we used a previously engineered genetically modified strain of the filamentous fungus Aspergillus nidulans, which contains the human estrogen receptor alpha and a reporter construct, in which β-galactosidase gene expression is controlled by an estrogen-responsive-element. The estrogen response of the fungal biosensor was validated with blood, urine, feces, milk and saliva. All matrices were screened for estrogenic activity prior to and after chemical extraction and the results were compared to an enzyme immunoassay (EIA). The biosensor showed consistent results in milk, urine and feces, which were comparable to those of the EIA. In contrast to the EIA, no sample pre-treatment by chemical extraction was needed. For 17β-estradiol, the biosensor showed a limit of detection of 1ng/L. The validation of the biosensor for pregnancy diagnosis revealed a specificity of 100% and a sensitivity of more than 97%. In conclusion, we developed and validated a highly robust fungal biosensor for detection of estrogen activity, which is highly sensitive and economic as it allows analyzing in high-throughput formats without the necessity for organic solvents.

Role of Aspergillus niger acrA in Arsenic Resistance and Its Use as the Basis for an Arsenic Biosensor

Arsenic contamination of groundwater sources is a major issue worldwide, since exposure to high levels of arsenic has been linked to a variety of health problems. Effective methods of detection are thus greatly needed as preventive measures. In an effort to develop a fungal biosensor for arsenic, we first identified seven putative arsenic metabolism and transport genes in Aspergillus niger, a widely used industrial organism that is generally regarded as safe (GRAS). Among the genes tested for RNA expression in response to arsenate, acrA, encoding a putative plasma membrane arsenite efflux pump, displayed an over 200-fold increase in gene expression in response to arsenate. We characterized the function of this A. niger protein in arsenic efflux by gene knockout and confirmed that AcrA was located at the cell membrane using an enhanced green fluorescent protein (eGFP) fusion construct. Based on our observations, we developed a putative biosensor strain containing a construct of the native promoter of acrA fused with egfp. We analyzed the fluorescence of this biosensor strain in the presence of arsenic using confocal microscopy and spectrofluorimetry. The biosensor strain reliably detected both arsenite and arsenate in the range of 1.8 to 180 μg/liter, which encompasses the threshold concentrations for drinking water set by the World Health Organization (10 and 50 μg/liter).

Development of a Fungal Biosensor for Field Verifying the Surface Disinfection of Ultraviolet Germicidal Irradiation Systems for Air Handling Units

This study developed a fungal biosensor to verify the germicidal effect of ultraviolet germicidal irradiation (UVGI) systems in the field from a biosensor originally developed to assess indoor climate. A UVC ray was irradiated on the fungal spores of the biosensors at various doses, and the differences in hyphal length after subsequent incubation were observed to verify the susceptibility of the fungal biosensor to C-band ultraviolet (UVC). The results showed reasonable correlations between UVC dosage and hyphal length. These fungal biosensors were thereafter attached to the coil and the drain pan of an air handling unit with a UVGI system installed and exposed to UVC for 1 h to test the effectiveness of the biosensors on the UVGI systems in the field. The hyphal lengths observed after incubation corresponded with UVC doses of 468–1620 J·m−2 at each sampling point as shown in the previous susceptibility experiments. The extended experiments to confirm the susceptibility of the fungal spores showed that the spores were more sensitive to UVC ray than those of the fungal biosensor and had similar susceptibility to the same UVC doses even at different UVC intensities.

Biosensing of an indoor volatile organic compound on the basis of fungal growth

A fungal biosensor plate was applied to assessment of the harmfulness of air polluted by formaldehyde. Alternaria alternata, Eurotium herbariorum and Aspergillus penicillioides were used as fungal species. Fungal mycelium length and optical transparency of the biosensor plate were employed as indices of the fungal growth. Formaldehyde in air was detected on the basis of growth inhibition, reflected by suppression of the growth indices. Dynamic range of the measurement was 700–4000 μg m −3 or broader. Eurotium herbariorum and Aspergillus penicillioides were the most suitable fungal species to formaldehyde sensing based on the mycelium length and that based on the transparency, respectively.

Impact of heavy metal amended sewage sludge on forest soils as assessed by bacterial and fungal biosensors

Bacterial and fungal bioluminescence-based biosensors were used as indicators of potential heavy metal toxicity to microorganisms in the needle litter of a mature Pinus radiata forest under heavy metal contaminated sewage sludge. Sewage sludge was amended with increasing concentrations of Cu, Ni and Zn and applied to the surface of a mature P. radiata forest. The response of the bacterial and fungal biosensors to soluble Cu, Ni and Zn in needle litter extracts was investigated. The bioluminescence response of the bacterial biosensor Escherichia coli HB101 pUCD607 declined as water-soluble Zn concentrations increased. The effective concentrations that gave a 50% reduction in bioluminescence (EC50 values) for water-soluble Zn and total litter Zn were 1.3 mg l−1 and 3700 mg kg−1, respectively. The bioluminescence response of the fungal biosensor Armillaria mellea declined as soluble Cu concentrations increased. The EC50 values for water-soluble Cu and total litter Cu were 0.12 mg l−1 and 540 mg kg−1, respectively. No decline in bioluminescence was noted for either the bacterial or fungal biosensor on exposure to increasing concentrations of water-soluble Ni. The use of a combination of bacterial and fungal biosensors offers a rapid and sensitive tool for assessing toxicity of heavy metals to microorganisms and, thus, elucidating the environmental impact of contaminants in sewage sludge on litter dwelling microorganisms.