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18 pages, 3877 KiB

Open AccessArticle

Batch Fabrication of Microelectrode Arrays with Glassy Carbon Microelectrodes and Interconnections for Neurochemical Sensing: Promises and Challenges

by Emma-Bernadette A. Faul, Austin M. Broussard, Daniel R. Rivera, May Yoon Pwint, Bingchen Wu, Qun Cao, Davis Bailey, X. Tracy Cui and Elisa Castagnola

Micromachines 2024, 15(2), 277; https://doi.org/10.3390/mi15020277 - 15 Feb 2024

Cited by 2 | Viewed by 1894

Abstract

Flexible multielectrode arrays with glassy carbon (GC) electrodes and metal interconnection (hybrid MEAs) have shown promising performance in multi-channel neurochemical sensing. A primary challenge faced by hybrid MEAs fabrication is the adhesion of the metal traces with the GC electrodes, as prolonged [...] Read more.

Flexible multielectrode arrays with glassy carbon (GC) electrodes and metal interconnection (hybrid MEAs) have shown promising performance in multi-channel neurochemical sensing. A primary challenge faced by hybrid MEAs fabrication is the adhesion of the metal traces with the GC electrodes, as prolonged electrical and mechanical stimulation can lead to adhesion failure. Previous devices with GC electrodes and interconnects made of a homogeneous material (all GC) demonstrated exceptional electrochemical stability but required miniaturization for enhanced tissue integration and chronic electrochemical sensing. In this study, we used two different methods for the fabrication of all GC-MEAs on thin flexible substrates with miniaturized features. The first method, like that previously reported, involves a double pattern-transfer photolithographic process, including transfer-bonding on temporary polymeric support. The second method requires a double-etching process, which uses a 2 µm-thick low stress silicon nitride coating of the Si wafer as the bottom insulator layer for the MEAs, bypassing the pattern-transfer and demonstrating a novel technique with potential advantages. We confirmed the feasibility of the two fabrication processes by verifying the practical conductivity of 3 µm-wide 2 µm-thick GC traces, the GC microelectrode functionality, and their sensing capability for the detection of serotonin using fast scan cyclic voltammetry. Through the exchange and discussion of insights regarding the strengths and limitations of these microfabrication methods, our goal is to propel the advancement of GC-based MEAs for the next generation of neural interface devices. Full article

(This article belongs to the Special Issue Biosensors for Biomedical and Environmental Applications, Volume 2)

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19 pages, 4477 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Laser-Induced Intracellular Delivery: Exploiting Gold-Coated Spiky Polymeric Nanoparticles and Gold Nanorods under Near-Infrared Pulses for Single-Cell Nano-Photon-Poration

by Ashish Kumar, Bishal Kumar Nahak, Pallavi Gupta, Tuhin Subhra Santra and Fan-Gang Tseng

Micromachines 2024, 15(2), 168; https://doi.org/10.3390/mi15020168 - 23 Jan 2024

Cited by 2 | Viewed by 1898

Abstract

This study explores the potential of laser-induced nano-photon-poration as a non-invasive technique for the intracellular delivery of micro/macromolecules at the single-cell level. This research proposes the utilization of gold-coated spiky polymeric nanoparticles (Au-PNPs) and gold nanorods (GNRs) to achieve efficient intracellular micro/macromolecule delivery [...] Read more.

This study explores the potential of laser-induced nano-photon-poration as a non-invasive technique for the intracellular delivery of micro/macromolecules at the single-cell level. This research proposes the utilization of gold-coated spiky polymeric nanoparticles (Au-PNPs) and gold nanorods (GNRs) to achieve efficient intracellular micro/macromolecule delivery at the single-cell level. By shifting the operating wavelength towards the near-infrared (NIR) range, the intracellular delivery efficiency and viability of Au-PNP-mediated photon-poration are compared to those using GNR-mediated intracellular delivery. Employing Au-PNPs as mediators in conjunction with nanosecond-pulsed lasers, a highly efficient intracellular delivery, while preserving high cell viability, is demonstrated. Laser pulses directed at Au-PNPs generate over a hundred hot spots per particle through plasmon resonance, facilitating the formation of photothermal vapor nanobubbles (PVNBs). These PVNBs create transient pores, enabling the gentle transfer of cargo from the extracellular to the intracellular milieu, without inducing deleterious effects in the cells. The optimization of wavelengths in the NIR region, coupled with low laser fluence (27 mJ/cm²) and nanoparticle concentrations (34 µg/mL), achieves outstanding delivery efficiencies (96%) and maintains high cell viability (up to 99%) across the various cell types, including cancer and neuronal cells. Importantly, sustained high cell viability (90–95%) is observed even 48 h post laser exposure. This innovative development holds considerable promise for diverse applications, encompassing drug delivery, gene therapy, and regenerative medicine. This study underscores the efficiency and versatility of the proposed technique, positioning it as a valuable tool for advancing intracellular delivery strategies in biomedical applications. Full article

(This article belongs to the Special Issue Biosensors for Biomedical and Environmental Applications, Volume 2)

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23 pages, 7656 KiB

Open AccessCommunication

A Hybrid Energy-Efficient, Area-Efficient, Low-Complexity Switching Scheme in SAR ADC for Biosensor Applications

by Yunfeng Hu, Chaoyi Chen, Qingming Huang, Lexing Hu, Bin Tang, Mengsi Hu, Bingbing Yuan, Zhaohui Wu and Bin Li

Micromachines 2024, 15(1), 60; https://doi.org/10.3390/mi15010060 - 27 Dec 2023

Cited by 2 | Viewed by 1272

Abstract

A hybrid energy-efficient, area-efficient, low-complexity switching scheme in SAR ADC for biosensor applications is proposed. This scheme is a combination of the monotonic technique, the MSB capacitor-splitting technique, and a new switching method. The MSB capacitor-splitting technique, as well as the reference voltage [...] Read more.

A hybrid energy-efficient, area-efficient, low-complexity switching scheme in SAR ADC for biosensor applications is proposed. This scheme is a combination of the monotonic technique, the MSB capacitor-splitting technique, and a new switching method. The MSB capacitor-splitting technique, as well as the reference voltage V_aq allow for more options for reference voltage conversion, resulting in higher area savings and higher energy efficiency. In a capacitor array, the circuit performs unilateral switching during all comparisons except for the second and last two comparisons, reducing the difficulty in designing the drive circuit. The proposed switching scheme saves 98.4% of the switching energy and reduces the number of unit capacitors by 87.5% compared to a conventional scheme. Furthermore, the SAR ADC employs low-noise and low-power dynamic comparators utilizing multi-clock control, low-sampling error-sampling switches based on the bootstrap technique, and dynamic SAR logic. The simulation results demonstrated that the proposed SAR ADC achieves 61.51 dB SNDR, 79.21 dB SFDR and consumes 0.278 μW of power in a 180 nm process with a 1 V power supply, a full swing input signal frequency of 23.33 kHz, and a sampling rate of 100 kS/s. Full article

(This article belongs to the Special Issue Biosensors for Biomedical and Environmental Applications, Volume 2)

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18 pages, 8792 KiB

Open AccessArticle

A Low-Power SAR ADC with Capacitor-Splitting Energy-Efficient Switching Scheme for Wearable Biosensor Applications

by Yunfeng Hu, Qingming Huang, Bin Tang, Chaoyi Chen, Lexing Hu, Enhao Yu, Bin Li and Zhaohui Wu

Micromachines 2023, 14(12), 2244; https://doi.org/10.3390/mi14122244 - 15 Dec 2023

Cited by 1 | Viewed by 1796

Abstract

A low-power SAR ADC with capacitor-splitting energy-efficient switching scheme is proposed for wearable biosensor applications. Based on capacitor-splitting, additional reference voltage V_cm, and common-mode techniques, the proposed switching scheme achieves 93.76% less switching energy compared to the conventional scheme with common-mode [...] Read more.

A low-power SAR ADC with capacitor-splitting energy-efficient switching scheme is proposed for wearable biosensor applications. Based on capacitor-splitting, additional reference voltage V_cm, and common-mode techniques, the proposed switching scheme achieves 93.76% less switching energy compared to the conventional scheme with common-mode voltage shift in one LSB. With the switching scheme, the proposed SAR ADC can lower the dependency on the accuracy of V_cm and the complexity of digital control logic and DAC driver circuits. Furthermore, the SAR ADC employs low-noise and low-power dynamic comparators utilizing multi-clock control, low sampling error sampling switches based on the bootstrap technique, and dynamic SAR logic. The simulation results demonstrate that the ADC achieves a 61.77 dB SNDR and a 78.06 dB SFDR and consumes 4.45 μW of power in a 180 nm process with a 1 V power supply, a full-swing input signal frequency of 93.33 kHz, and a sampling rate of 200 kS/s. Full article

(This article belongs to the Special Issue Biosensors for Biomedical and Environmental Applications, Volume 2)

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13 pages, 4479 KiB

Open AccessArticle

Espial: Electrochemical Soil pH Sensor for In Situ Real-Time Monitoring

by Mohammed A. Eldeeb, Vikram Narayanan Dhamu, Anirban Paul, Sriram Muthukumar and Shalini Prasad

Micromachines 2023, 14(12), 2188; https://doi.org/10.3390/mi14122188 - 30 Nov 2023

Cited by 3 | Viewed by 2105

Abstract

We present a first-of-its-kind electrochemical sensor that demonstrates direct real-time continuous soil pH measurement without any soil pre-treatment. The sensor functionality, performance, and in-soil dynamics have been reported. The sensor coating is a composite matrix of alizarin and Nafion applied by drop casting [...] Read more.

We present a first-of-its-kind electrochemical sensor that demonstrates direct real-time continuous soil pH measurement without any soil pre-treatment. The sensor functionality, performance, and in-soil dynamics have been reported. The sensor coating is a composite matrix of alizarin and Nafion applied by drop casting onto the working electrode. Electrochemical impedance spectroscopy (EIS) and squarewave voltammetry (SWV) studies were conducted to demonstrate the functionality of each method in accurately detecting soil pH. The studies were conducted on three different soil textures (clay, sandy loam, and loamy clay) to cover the range of the soil texture triangle. Squarewave voltammetry showed pH-dependent responses regardless of soil texture (while electrochemical impedance spectroscopy’s pH detection range was limited and dependent on soil texture). The linear models showed a sensitivity range from −50 mV/pH up to −66 mV/pH with R² > 0.97 for the various soil textures in the pH range 3–9. The validation of the sensor showed less than a 10% error rate between the measured pH and reference pH for multiple different soil textures including ones that were not used in the calibration of the sensor. A 7-day in situ soil study showed the capability of the sensor to measure soil pH in a temporally dynamic manner with an error rate of less than 10%. The test was conducted using acidic and alkaline soils with pH values of 5.05 and 8.36, respectively. Full article

(This article belongs to the Special Issue Biosensors for Biomedical and Environmental Applications, Volume 2)

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17 pages, 4940 KiB

Open AccessArticle

A Multi-Layer Breast Cancer Model to Study the Synergistic Effect of Photochemotherapy

by Magdalena Flont and Elżbieta Jastrzębska

Micromachines 2023, 14(9), 1806; https://doi.org/10.3390/mi14091806 - 21 Sep 2023

Viewed by 1399

Abstract

Breast cancer is one of the most common cancers among women. The development of new and effective therapeutic approaches in the treatment of breast cancer is an important challenge in modern oncology. Two-dimensional (2D) cell cultures are most often used in the study [...] Read more.

Breast cancer is one of the most common cancers among women. The development of new and effective therapeutic approaches in the treatment of breast cancer is an important challenge in modern oncology. Two-dimensional (2D) cell cultures are most often used in the study of compounds with potential anti-tumor nature. However, it is necessary to develop advanced three-dimensional (3D) cell models that can, to some extent, reflect the physiological conditions. The use of miniature cancer-on-a-chip microfluidic systems can help to mimic the complex cancer microenvironment. In this report, we developed a 3D breast cancer model in the form of a cell multilayer, composed of stromal cells (HMF) and breast cancer parenchyma (MCF-7). The developed cell model was successfully used to analyze the effectiveness of combined sequential photochemotherapy, based on doxorubicin and meso-tetraphenylporphyrin. We proved that the key factor that allows achieving the synergistic effect of combination therapy are the order of drug administration to the cells and the sequence of therapeutic procedures. To the best of our knowledge, studies on the effectiveness of combination photochemotherapy depending on the sequence of the component drugs were performed for the first time under microfluidic conditions on a 3D multilayered model of breast cancer tissue. Full article

(This article belongs to the Special Issue Biosensors for Biomedical and Environmental Applications, Volume 2)

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19 pages, 9593 KiB

Open AccessArticle

Thin Films of Chlorinated Vanadyl Phthalocyanines as Active Layers of Chemiresistive Sensors for the Detection of Ammonia

by Darya Klyamer, Alexandr Sukhikh, Dmitry Bonegardt, Pavel Krasnov, Pavel Popovetskiy and Tamara Basova

Micromachines 2023, 14(9), 1773; https://doi.org/10.3390/mi14091773 - 15 Sep 2023

Cited by 2 | Viewed by 1135

Abstract

Halogenated metal phthalocyanines are promising materials for the manufacture of active layers of chemiresistive sensors for the detection of various gases. Despite the high interest in such sensors, there are few systematic studies of the position of halogen substituents in phthalocyanine macroring on [...] Read more.

Halogenated metal phthalocyanines are promising materials for the manufacture of active layers of chemiresistive sensors for the detection of various gases. Despite the high interest in such sensors, there are few systematic studies of the position of halogen substituents in phthalocyanine macroring on the chemiresistive response of their films to gases. In this work, we prepared and studied films of novel tetrachlorosubstituted vanadyl phthalocyanine derivatives with Cl substituents in the peripheral (VOPcCl₄-p) and nonperipheral (VOPcCl₄-np) positions of the phthalocyanine ring as active layers of chemiresistive sensors to reveal the effect of the position of substituents on their structure and sensor response to low concentrations of NH₃. It was shown that the films of VOPcCl₄-p exhibited a noticeably higher sensor response to NH₃ than the VOPcCl₄-np ones. The limit of detection of NH₃ was 0.7 ppm. The sensing layers demonstrated a reversible sensor response at room temperature with fairly low response/recovery times. It was also demonstrated that NH₃ can be detected in the presence of various interfering gases (CO₂ and H₂) and some volatile organic vapors, as well as in a mixture of gases with a composition close to exhaled air. Full article

(This article belongs to the Special Issue Biosensors for Biomedical and Environmental Applications, Volume 2)

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10 pages, 1684 KiB

Open AccessArticle

Mode Characterization and Sensitivity Evaluation of a Surface Acoustic Wave (SAW) Resonator Biosensor: Application to the Glial-Fibrillary-Acidic-Protein (GFAP) Biomarker Detection

by Antonio Matteo Passeri, Francesco Lunardelli, Daniele Cavariani, Marco Cecchini and Matteo Agostini

Micromachines 2023, 14(8), 1485; https://doi.org/10.3390/mi14081485 - 25 Jul 2023

Viewed by 1272

Abstract

Biosensors based on surface acoustic waves (SAWs) offer unique advantages due to their high sensitivity, real-time response capability, and label-free detection. The typical SAW modes are the Rayleigh mode and the shear-horizontal mode. Both present pros and cons for biosensing applications and generally [...] Read more.

Biosensors based on surface acoustic waves (SAWs) offer unique advantages due to their high sensitivity, real-time response capability, and label-free detection. The typical SAW modes are the Rayleigh mode and the shear-horizontal mode. Both present pros and cons for biosensing applications and generally need different substrates and device geometries to be efficiently generated. This study investigates and characterizes SAW resonator biosensors on lithium niobate in terms of modes generated and biosensing performance. It reveals the simultaneous presence of two typical SAW modes, the first around 1.6 GHz and the second around 1.9 GHz, differently polarized and clearly separated in frequency, which we refer to as slow and fast modes. The two modes are studied by numerical simulations and biosensing experiments with the glial-fibrillary-acidic-protein (GFAP) biomarker. The slow mode is generally more sensitive to changes in surface properties, such as temperature and mass changes, by a factor of about 1.4 with respect to the fast mode. Full article

(This article belongs to the Special Issue Biosensors for Biomedical and Environmental Applications, Volume 2)

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Review

Jump to: Research

32 pages, 2887 KiB

Open AccessReview

A Topical Review on Enabling Technologies for the Internet of Medical Things: Sensors, Devices, Platforms, and Applications

by Md. Shamsul Arefin, Mohammed Mostafizur Rahman, Md. Tanvir Hasan and Mufti Mahmud

Micromachines 2024, 15(4), 479; https://doi.org/10.3390/mi15040479 - 30 Mar 2024

Viewed by 1988

Abstract

The Internet of Things (IoT) is still a relatively new field of research, and its potential to be used in the healthcare and medical sectors is enormous. In the last five years, IoT has been a go-to option for various applications such as [...] Read more.

The Internet of Things (IoT) is still a relatively new field of research, and its potential to be used in the healthcare and medical sectors is enormous. In the last five years, IoT has been a go-to option for various applications such as using sensors for different features, machine-to-machine communication, etc., but precisely in the medical sector, it is still lagging far behind compared to other sectors. Hence, this study emphasises IoT applications in medical fields, Medical IoT sensors and devices, IoT platforms for data visualisation, and artificial intelligence in medical applications. A systematic review considering PRISMA guidelines on research articles as well as the websites on IoMT sensors and devices has been carried out. After the year 2001, an integrated outcome of 986 articles was initially selected, and by applying the inclusion–exclusion criterion, a total of 597 articles were identified. 23 new studies have been finally found, including records from websites and citations. This review then analyses different sensor monitoring circuits in detail, considering an Intensive Care Unit (ICU) scenario, device applications, and the data management system, including IoT platforms for the patients. Lastly, detailed discussion and challenges have been outlined, and possible prospects have been presented. Full article

(This article belongs to the Special Issue Biosensors for Biomedical and Environmental Applications, Volume 2)

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18 pages, 2360 KiB

Open AccessReview

Enzymes Immobilized into Starch- and Gelatin-Based Hydrogels: Properties and Application in Inhibition Assay

by Elena N. Esimbekova, Irina G. Torgashina, Elena V. Nemtseva and Valentina A. Kratasyuk

Micromachines 2023, 14(12), 2217; https://doi.org/10.3390/mi14122217 - 8 Dec 2023

Cited by 1 | Viewed by 1781

Abstract

The present work is a review of the research on using hydrogels based on natural biodegradable polymers, starch, and gelatin for enzyme immobilization. This review addresses the main properties of starch and gelatin that make them promising materials in biotechnology for producing enzyme [...] Read more.

The present work is a review of the research on using hydrogels based on natural biodegradable polymers, starch, and gelatin for enzyme immobilization. This review addresses the main properties of starch and gelatin that make them promising materials in biotechnology for producing enzyme preparations stable during use and storage and insensitive to chemical and physical impacts. The authors summarize their achievements in developing the preparations of enzymes immobilized in starch and gelatin gels and assess their activity, stability, and sensitivity for use as biorecognition elements of enzyme inhibition-based biosensors. Full article

(This article belongs to the Special Issue Biosensors for Biomedical and Environmental Applications, Volume 2)

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24 pages, 4980 KiB

Open AccessReview

Titanium Carbide (Ti₃C₂T_x) MXene as Efficient Electron/Hole Transport Material for Perovskite Solar Cells and Electrode Material for Electrochemical Biosensors/Non-Biosensors Applications

by Theophile Niyitanga, Archana Chaudhary, Khursheed Ahmad and Haekyoung Kim

Micromachines 2023, 14(10), 1907; https://doi.org/10.3390/mi14101907 - 6 Oct 2023

Cited by 8 | Viewed by 2082

Abstract

Recently, two-dimensional (2D) MXenes materials have received enormous attention because of their excellent physiochemical properties such as high carrier mobility, metallic electrical conductivity, mechanical properties, transparency, and tunable work function. MXenes play a significant role as additives, charge transfer layers, and conductive electrodes [...] Read more.

Recently, two-dimensional (2D) MXenes materials have received enormous attention because of their excellent physiochemical properties such as high carrier mobility, metallic electrical conductivity, mechanical properties, transparency, and tunable work function. MXenes play a significant role as additives, charge transfer layers, and conductive electrodes for optoelectronic applications. Particularly, titanium carbide (Ti₃C₂T_x) MXene demonstrates excellent optoelectronic features, tunable work function, good electron affinity, and high conductivity. The Ti₃C₂T_x has been widely used as electron transport (ETL) or hole transport layers (HTL) in the development of perovskite solar cells (PSCs). Additionally, Ti₃C₂T_x has excellent electrochemical properties and has been widely explored as sensing material for the development of electrochemical biosensors. In this review article, we have summarized the recent advances in the development of the PSCs using Ti₃C₂T_x MXene as ETL and HTL. We have also compiled the recent progress in the fabrication of biosensors using Ti₃C₂T_x-based electrode materials. We believed that the present mini review article would be useful to provide a deep understanding, and comprehensive insight into the research status. Full article

(This article belongs to the Special Issue Biosensors for Biomedical and Environmental Applications, Volume 2)

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13 pages, 1904 KiB

Open AccessReview

Advancements in SARS-CoV-2 Testing: Enhancing Accessibility through Machine Learning-Enhanced Biosensors

by Antonios Georgas, Konstantinos Georgas and Evangelos Hristoforou

Micromachines 2023, 14(8), 1518; https://doi.org/10.3390/mi14081518 - 28 Jul 2023

Cited by 1 | Viewed by 1337

Abstract

The COVID-19 pandemic highlighted the importance of widespread testing for SARS-CoV-2, leading to the development of various new testing methods. However, traditional invasive sampling methods can be uncomfortable and even painful, creating barriers to testing accessibility. In this article, we explore how machine [...] Read more.

The COVID-19 pandemic highlighted the importance of widespread testing for SARS-CoV-2, leading to the development of various new testing methods. However, traditional invasive sampling methods can be uncomfortable and even painful, creating barriers to testing accessibility. In this article, we explore how machine learning-enhanced biosensors can enable non-invasive sampling for SARS-CoV-2 testing, revolutionizing the way we detect and monitor the virus. By detecting and measuring specific biomarkers in body fluids or other samples, these biosensors can provide accurate and accessible testing options that do not require invasive procedures. We provide examples of how these biosensors can be used for non-invasive SARS-CoV-2 testing, such as saliva-based testing. We also discuss the potential impact of non-invasive testing on accessibility and accuracy of testing. Finally, we discuss potential limitations or biases associated with the machine learning algorithms used to improve the biosensors and explore future directions in the field of machine learning-enhanced biosensors for SARS-CoV-2 testing, considering their potential impact on global healthcare and disease control. Full article

(This article belongs to the Special Issue Biosensors for Biomedical and Environmental Applications, Volume 2)

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15 pages, 1967 KiB

Open AccessReview

Ultrasensitive Electrochemical Immunosensors Using Nanobodies as Biocompatible Sniffer Tools of Agricultural Contaminants and Human Disease Biomarkers

by Rodica Elena Ionescu

Micromachines 2023, 14(8), 1486; https://doi.org/10.3390/mi14081486 - 25 Jul 2023

Viewed by 2209

Abstract

Nanobodies (Nbs) are known as camelid single-domain fragments or variable heavy chain antibodies (VHH) that in vitro recognize the antigens (Ag) similar to full-size antibodies (Abs) and in vivo allow immunoreactions with biomolecule cavities inaccessible to conventional Abs. Currently, Nbs are widely used [...] Read more.

Nanobodies (Nbs) are known as camelid single-domain fragments or variable heavy chain antibodies (VHH) that in vitro recognize the antigens (Ag) similar to full-size antibodies (Abs) and in vivo allow immunoreactions with biomolecule cavities inaccessible to conventional Abs. Currently, Nbs are widely used for clinical treatments due to their remarkably improved performance, ease of production, thermal robustness, superior physical and chemical properties. Interestingly, Nbs are also very promising bioreceptors for future rapid and portable immunoassays, compared to those using unstable full-size antibodies. For all these reasons, Nbs are excellent candidates in ecological risk assessments and advanced medicine, enabling the development of ultrasensitive biosensing platforms. In this review, immobilization strategies of Nbs on conductive supports for enhanced electrochemical immune detection of food contaminants (Fcont) and human biomarkers (Hbio) are discussed. In the case of Fcont, the direct competitive immunoassay detection using coating antigen solid surface is the most commonly used approach for efficient Nbs capture which was characterized with cyclic voltammetry (CV) and differential pulse voltammetry (DPV) when the signal decays for increasing concentrations of free antigen prepared in aqueous solutions. In contrast, for the Hbio investigations on thiolated gold electrodes, increases in amperometric and electrochemical impedance spectroscopy (EIS) signals were recorded, with increases in the antigen concentrations prepared in PBS or spiked real human samples. Full article

(This article belongs to the Special Issue Biosensors for Biomedical and Environmental Applications, Volume 2)

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