Capture Of Cancer Cells Research Articles

A dielectrophoresis-based platform of cancerous cells capture using aptamer-functionalized gold nanoparticles in a microfluidic channel.

In this paper, a microfluidic chip for the manipulation and capture of cancer cells was introduce. In which, the combination of dielectrophoresis (DEP) and a binding method based on chemical interactions by using cell-specific aptamer was performed to enhance the capture strength and specificity. The device has been simply constructed from a straight-channel PDMS placed on a glass substrate that patterned electrode structures and a self-assembled monolayer (SAM) of gold nanoparticles (AuNPs). The target cells were transported to the manipulation area by flow and attracted down to the region between the electrodes under the influence of positive dielectrophoresis force (pDEP). This approach facilitated subsequent selective capture by the modified aptamers on the AuNPs. The distribution of the electric field in the channel has also been simulated to clarify the DEP operation. As a result, the device has been shown to effectively capture target lung cancer cells with a concentration as low as cells/mL. The capture specificity in a sample of mixed cells is up to 80.4%. This technique has the potential to be applied to detection methods for many types of cancer. This article is protected by copyright. All rights reserved.

Targeting Mucin Protein Enables Rapid and Efficient Ovarian Cancer Cell Capture: Role of Nanoparticle Properties in Efficient Capture and Culture.

The development of specific and sensitive immunomagnetic cell separation nanotechnologies is central to enhancing the diagnostic relevance of circulating tumor cells (CTCs) and improving cancer patient outcomes. The limited number of specific biomarkers used to enrich a phenotypically diverse set of CTCs from liquid biopsies has limited CTC yields and purity. The ultra-high molecular weight mucin, mucin16(MUC16) is shown to physically shield key membrane proteins responsible for activating immune responses against ovarian cancer cells and may interfere with the binding of magnetic nanoparticles to popular immunomagnetic cell capture antigens. MUC16 is expressed in ≈90% of ovarian cancers and is almost universal in High Grade Serous Epithelial Ovarian Cancer. This work demonstrates that cell bound MUC16 is an effective target for rapid immunomagnetic extraction of expressor cells with near quantitative yield, high purity and viability from serum. The results provide a mechanistic insight into the effects of nanoparticle physical properties and immunomagnetic labeling on the efficiency of immunomagnetic cell isolation. The growth of these cells has also been studied after separation, demonstrating that nanoparticle size impacts cell-particle behavior and growth rate. These results present the successful isolation of "masked" CTCs enabling new strategies for the detection of cancer recurrence and select and monitor chemotherapy.

The detection of the captured circulating tumor cells on the core-shell nanofibrous membrane using hyaluronic acid-functionalized graphene quantum dots.

In recent years, cancerous cases have increased remarkably worldwide, and metastasis is the leading cause of death. Therefore, research on the early detection of cancer and metastasis has expanded to aid successful cancer treatment. Here in this paper, at the first step, an electrospun nanofibrous membrane (NFM) with a core-shell structure was fabricated from PCL and HA to achieve cancer cell capturing (about 75% of cells). On the other hand, hyaluronic acid (HA)-functionalized graphene quantum dots (GQDs) were used to detect captured cancer cells on NFM through the changes in photoluminescence intensity. Therefore, CD44 receptor-HA interaction is the main principle used for both entrapment and detection of cancer cells. Results demonstrated the GQD-HA fluorescent intensity of solution decreased through the increase of the captured cancer cell numbers on NFM, which is related to the more adsorption of GQD nanocomposites to the CD44 receptors. In contrast, this intensity for noncancerous cells was steady with any cell concentrations. This difference shows the system's remarkable selectivity and specificity, which can be crucial in fluorescent imaging for accurate cancer diagnosis.

Capture and Release of Cancer Cells Through Smart Bioelectronics.

Noninvasive collection of target cells such as circulating tumor cells (CTCs) is crucial for biology and medicine research. Conventional methods of cell collection are often complex, requiring either size-dependent sorting or invasive enzymatic reactions. Here, we show the development of a functional polymer film, which combines the thermoresponsive poly(N-isopropylacrylamide) and the conducting poly(3,4-ethylenedioxythiopene)/poly(styrene sulfonate), and its use for the capture and release of CTCs. When coated onto microfabricated gold electrodes, the proposed polymer films are capable of noninvasively capturing and controllably releasing cells while, at the same time, monitoring these processes with conventional electrical measurements.

One-dimensional nanomaterials for cancer therapy and diagnosis.

One-dimensional (1-D) nanomaterials possess unique shape-dependent phyicochemical properties and are increasingly recognized as promising materials for nanotechnology. 1-D nanomaterials can be classified according to their shape, such as nanorods, nanotubes, nanowires, self-assembled nanochains, etc., and have been applied in electronics, photonics, and catalysis. The biological characteristics of 1-D nanomaterials, including high drug loading efficiency, prolonged blood circulation, the ability to capture cancer cells, unique cellular uptake mechanisms, efficient photothermal conversion, and material tunability, have aided in extending their potential to biomedical applications, particularly in cancer therapy and diagnosis. This review highlights a novel perspective on emerging 1-D nanomaterials for cancer therapy and diagnosis by introducing the definition of 1-D nanomaterials, their shape-dependent physicochemical properties, biomedical applications, and recent advances in cancer therapy and diagnosis. This review also proposes unexplored potential nanomaterial types and therapeutic applications for 1-D nanomaterials. In particular, the most significant and exciting advances in recent years, including ultrasound-enabled sonodynamic therapy, magnetic field-based therapy, and bioresponsive 1-D nanomaterials for intracellular self-assembly in situ, are discussed along with novel therapeutic concepts, such as piezoelectric 1-D nanomaterials, nanozyme-based nanomedicine, and others.

MyCTC chip: microfluidic-based drug screen with patient-derived tumour cells from liquid biopsies

Cancer patients with advanced disease are characterized by intrinsic challenges in predicting drug response patterns, often leading to ineffective treatment. Current clinical practice for treatment decision-making is commonly based on primary or secondary tumour biopsies, yet when disease progression accelerates, tissue biopsies are not performed on a regular basis. It is in this context that liquid biopsies may offer a unique window to uncover key vulnerabilities, providing valuable information about previously underappreciated treatment opportunities. Here, we present MyCTC chip, a novel microfluidic device enabling the isolation, culture and drug susceptibility testing of cancer cells derived from liquid biopsies. Cancer cell capture is achieved through a label-free, antigen-agnostic enrichment method, and it is followed by cultivation in dedicated conditions, allowing on-chip expansion of captured cells. Upon growth, cancer cells are then transferred to drug screen chambers located within the same device, where multiple compounds can be tested simultaneously. We demonstrate MyCTC chip performance by means of spike-in experiments with patient-derived breast circulating tumour cells, enabling >95% capture rates, as well as prospective processing of blood from breast cancer patients and ascites fluid from patients with ovarian, tubal and endometrial cancer, where sensitivity to specific chemotherapeutic agents was identified. Together, we provide evidence that MyCTC chip may be used to identify personalized drug response patterns in patients with advanced metastatic disease and with limited treatment opportunities.

Functionalized Graphitic Carbon Nitride Nanomaterials for Electrochemiluminescent Detection of Cancer Cells

Biosensors capable of detecting cancer cell quantity is significant for cancer pathological investigation and prognosis. Herein, a facile electrochemiluminescent (ECL) biosensor is developed for cancer cell detection by comprehensively utilizing the features of functionalized graphitic carbon nitride (g-CN) nanomaterials like well film-forming capability and functionalization level tunability. The solid-state ECL biosensor is fabricated via a simple dropping-drying method by subsequent deposition of g-CN nanosheets/graphene oxide nanocomposites (CNNS/GO) as ECL emitter, folate functionalized g-CN quantum dots (FA-CNQDs) as cancer cell capture agent, and bull serum albumin (BSA) as blocker to prevent nonspecific binding on glassy carbon electrodes (GCEs). The strong and stable ECL emission of CNNS/GO along with the tunability of FA content in FA-CNQDs endow the ECL biosensor with competitive sensitivity, which is able to detect folate receptor-positive cancer cells (HepG2) in the concentration range of 102 − 104 cells ml−1 under optimal condition. Additionally, the proposed ECL biosensor shows high specificity, reproducibility and long-term stability as well as good reliability towards serum sample detection.

Uterine lavage identifies cancer mutations and increased TP53 somatic mutation burden in individuals with ovarian cancer.

Current screening methods for ovarian cancer (OC) have failed to demonstrate a significant reduction in mortality. Uterine lavage combined with TP53 ultra-deep sequencing for the detection of disseminated OC cells has emerged as a promising tool, but this approach has not been tested for early-stage disease or non-serous histologies. In addition, lavages carry multiple background mutations, the significance of which is poorly understood. Uterine lavage was collected preoperatively in 34 patients undergoing surgery for suspected ovarian malignancy including 14 patients with benign disease and 20 patients with OC (6 non-serous and 14 high grade serous-like (serous)). Ultra-deep duplex sequencing (~3000x) with a panel of common OC genes identified the tumor mutation in 33% of non-serous (all early stage) and in 79% of serous cancers (including four early stage). In addition, all lavages carried multiple somatic mutations (average of 25 mutations per lavage), more than half of which corresponded to common cancer driver mutations. Driver mutations in KRAS, PIK3CA, PTEN, PPP2R1A and ARID1A presented as larger clones than non-driver mutations and with similar frequency in lavages from patients with and without OC, indicating prevalent somatic evolution in all patients. Driver TP53 mutations, however, presented as significantly larger clones and with higher frequency in lavages from individuals with OC, suggesting that TP53-specific clonal expansions are linked to ovarian cancer development. Our results demonstrate that lavages capture cancer cells, even from early-stage cancers, as well as other clonal expansions and support further exploration of TP53 mutation burden as a potential OC risk factor.

Facile Preparation of Three-Dimensional Wafer with Interconnected Porous Structure for High-Performance Capture and Nondestructive Release of Circulating Tumor Cells.

Efficient isolation and downstream bioinformation analysis of circulating tumor cells (CTCs) in whole blood contribute to the early diagnosis of cancer and investigation of cancer metastasis. However, the separation and release of CTCs remain a great challenge due to the extreme rarity of CTCs and severe interference from other cells in complex clinical samples. Herein, we developed a low-cost and easy-to-fabricate aptamer-functionalized wafer with a three-dimensional (3D) interconnected porous structure by grafting polydopamine (PDA), poly(ethylene glycol) (PEG), and aptamer in sequence (Ni@PDA-PEG-Apt) for the capture and release of CTCs. The Ni@PDA-PEG-Apt wafer integrated the features of Ni foam with a 3D interconnected porous structure offering enough tunnels for cells to flow through and enhancing aptamer-cell contact frequency, the spacer PEG with flexible and high hydrophilic property increasing anti-interference ability and providing the wafer with more binding sites for aptamer, which result in an enhanced capture specificity and efficiency for CTCs. Because of these advantages, the Ni@PDA-PEG-Apt wafer achieved a high capture efficiency of 78.25%. The captured cancer cells were mildly released by endonuclease with up to 61.85% efficiency and good proliferation. Furthermore, tumor cells were injected into mice and experienced circulation in vivo. In blood samples after circulation, 65% of target tumor cells can be efficiently captured by the wafer, followed by released and recultured cells with high viability. Further downstream metabolomics analysis showed that target cancer cells remained with high biological activity and can be well separated from MCF-10A cells based on metabolic profiles by the PCA analysis, indicating the great potential of our strategy for further research on the progression of cancer metastasis. Notably, not only is the wafer cheap with a cost of only 3.58 U.S. dollars and easily prepared by environmental-friendly reagents but also the process of capturing and releasing tumor cells can be completed within an hour, which is beneficial for large-scale clinical use in the future.

Aptamer-Based Cancer Cell Analysis and Treatment.

Aptamers are a class of single‐stranded DNA or RNA oligonucleotides that can exclusively bind to various targets with high affinity and selectivity. Regarded as “chemical antibodies”, aptamers possess several intrinsic advantages, including easy synthesis, convenient modification, high programmability, and good biocompatibility. In recent decades, many studies have demonstrated the superiority of aptamers as molecular tools for various biological applications, particularly in the area of cancer theranostics. In this review, we focus on recent progress in developing aptamer‐based strategies for the precise analysis and treatment of cancer cells.

Lipid-mediated ex vivo cell surface engineering for augmented cellular functionalities.

Once administrated, intercellular adhesion to recognize and/or arrest target cells is essential for specific treatments, especially for cancer or tumor. However, immune cells administrated into the tumor-microenvironment could lose their intrinsic functionalities such as target recognition ability, resulting in an ineffective cancer immunotherapy. Various manipulation techniques for decorating functional moieties onto cell surface and enhancing target recognition have been developed. A hydrophobic interaction-mediated ex-vivo cell surface engineering using lipid-based biomaterials could be a state-of-the-art engineering technique that could achieve high-efficiency cell surface modification by a single method without disturbance of intrinsic characteristics of cells. In this regard, this review provides design principles for the development of lipid-based biomaterials with a linear structure of lipid, polyethylene glycol, and functional group, strategies for the synthesis process, and their practical applications in biomedical engineering. Especially, we provide new insights into the development of a novel surface coating techniques for natural killer (NK) cells with engineering decoration of cancer targeting moieties on their cell surfaces. Among immune cells, NK cells are interesting cell population for substituting T cells because of their excellent safety and independent anticancer efficacy. Thus, optimal strategies to select cancer-type-specific targeting moieties and present them onto the surface of immune cells (especially, NK cells) using lipid-based biomaterials could provide additional tools to capture cancer cells for developing novel immune cell therapy products. Enhanced anticancer efficacies by surface-engineered NK cells have been demonstrated both in vitro and in vivo. Therefore, it could be speculated that recent progresses in cell surface modification technology via lipid-based biomaterials could strengthen immune surveillance and immune synapses for utilization in a next-generation cancer immunotherapy, beyond currently available genetic engineering tool such as chimeric antigen receptor-mediated immune cell modulation.

Efficient Capture and Separation of Cancer Cells Using Hyaluronic Acid-Modified Magnetic Beads in a Microfluidic Chip.

The efficient isolation and specific discrimination of circulating tumor cells (CTCs) is expected to provide valuable information for understanding tumor metastasis and play an important role in the treatment of cancer patients. In this study, we developed a novel and rapid method for efficient capture and specific identification of cancer cells using hyaluronic acid (HA)-modified SiO2-coated magnetic beads in a microfluidic chip. First, polyacrylamide-surfaced SiO2-coated magnetic beads (SiO2@MBs) were covalently conjugated with HA, and the created HA-modified SiO2@MBs (HA-SiO2@MBs) display binding specificity to HeLa cells (a human cervical carcinoma cell line) overexpressing CD44 receptors. After incubating the HA-SiO2@MBs with cancer cells for 1 h, the mixture of MBs and cells was drawn into a designed microfluidic channel with two inlets and outlets. Through the formation of lamellar flow, cells specifically bound with the HA-SiO2@MBs can be separated under an external magnetic field with a capture efficiency of up to 92.0%. The developed method is simple, fast, and promising for CTC separation and cancer diagnostics applications.

An entropy-driven three-dimensional multipedal-DNA walker for ultrasensitive detection of cancer cells

Sensitive and accurate detection of cancer cells is of great significance for the early diagnosis and treatment of cancer. In this work, we developed a simple fluorescent signal amplification biosensor based on an entropy-driven three-dimensional (3D) multipedal-DNA walker for highly sensitive detection of cancer cells. Firstly, DNA tetrahedron nanostructures (DTNs) combined with AS1411 aptamer were used as the capture probe to achieve efficient capture of cancer cells. Then, the bipedal hairpin fuel chain hybridized with DTNs and exposed two catalytic “legs” to form a walker probe. Finally, the walker probe autonomously walked on polystyrene microspheres (PS) via entropy-driven catalytic reaction. DTNs rolled on the PS to achieve multipedal walking, realizing fluorescence signal amplification due to fluorescence recovery of DNA-CdTe quantum dots on the PS surface. This fluorescence signal amplification strategy showed excellent selectivity and sensitivity toward cancer cells with the detection limit of 7 cell mL−1. This entropy-driven 3D multipedal DNA walker fluorescence exhibited great potential in detecting circulating tumor cells and tumor markers used for early diagnosis and clinical treatment of cancer.

Effect of Chitosan on Alginate-Based Macroporous Hydrogels for the Capture of Glioblastoma Cancer Cells.

Glioblastoma multiforme is a type of brain cancer associated with a very low survival rate since a large number of cancer cells remain infiltrated in the brain despite the treatments currently available. This work presents a macroporous hydrogel trap, destined to be implanted in the surgical cavity following tumor resection and designed to attract and retain cancer cells, in order to eliminate them afterward with a lethal dose of stereotactic radiotherapy. The biocompatible hydrogel formulation comprises sodium alginate (SA) and chitosan (CHI) bearing complementary electrostatic charges and stabilizing the gels in saline and cell culture media, as compared to pristine SA gels. The highly controlled and interconnected porosity, characterized by X-ray microCT, yields mechanical properties comparable to those of brain tissues and allows F98 glioblastoma cells to penetrate the gels within the entire volume, as confirmed by fluorescence microscopy. The addition of a grafted -RGD peptide on SA, combined with CHI, significantly enhances the adhesion and retention of F98 cells within the gels. Overall, the best compromise between low proliferation and a high level of accumulation and retention of F98 cells was obtained with the hydrogel formulated with 1% SA and 0.2% CHI, without the -RGD adhesion peptide.

Characterization and Identification of Aptamers against CD49c for the Detection, Capture, and Release of Cancer Cells.

As a kind of recognition molecule, aptamers can be inserted into some regulatory sequences for the smart response of their targets. However, the molecular engineering might lead to the change of the binding affinity. Here, we present a stable aptamer ZAJ-2c and an environmentally sensitive aptamer ZAJ-2d optimized from an original cell-binding aptamer ZAJ-2, and the molecular target was further identified as CD49c on the cell membrane. ZAJ-2c was characterized with high binding ability independent of the presence of divalent cations at a temperature range from 4 to 37 °C, showing promise for measuring the expression of CD49c on cancer cells. Moreover, ZAJ-2d had a nanomolar binding affinity in the binding buffer at 4 °C, the same as ZAJ-2c, but lost the binding ability in a PBS buffer supplemented with 5 mM EDTA at 37 °C. This aptamer variant proved to selectively capture and release the CD49c positive cells by simply adjusting the temperatures and divalent cations. This set of aptamers might provide a toolbox for monitoring and operating of a wide range of cancer cells with CD49c expression on the surface, which will be helpful for the studying the heterogeneity of rare cells.

Extracorporeal microchannel device to capture and eliminate circulating tumor cells from cancer patient’s blood.

e14522 Background: Metastatic progression accounts for nearly 90% of cancer-related deaths and has been directly correlated with the presence of circulating tumor cells (CTCs) in numerous carcinomas including breast, lung, ovarian, colorectal, and head and neck cancers. The removal of CTCs from cancer patient’s blood is directly implicated with reduction of extravasation and disease invasiveness to secondary organs. Methods: We designed and printed 3 Dimensional (3D) microchannel devices using biocompatible polymer and packed it with anti-EpCAM (EpCAM) mediated glass-based (G) compositions (G-EpCAM). The computational fluid dynamic (CFD) analysis simulation was explored to optimize the hemodynamic effect of the G-EpCAM device for measuring the pressure and velocity difference for blood along the spiral flow microchannels. Red blood cell (RBC) hemolysis was estimated using G-EpCAM compositions packed in a device to determine optimal biocompatibility. We assessed cancer cell lines (breast cancer MCF7, lung cancer A549) interactions and capture with varying incubation time points, effect of anti-EpCAM concentrations, number of G-EpCAMs, and series of devices. We evaluated G-EpCAM-on-device’s CTC capture capability and biocompatibility using head and neck, colorectal, lung, and ductal breast cancer patient’s blood samples. All G-EpCAM captured CTCs were immuno-stained for cytokeratin 18 (CK18) expression and the optimal fluorescence acquisition intensity was quantified. Results: Extracorporeal G-EpCAM microchannel device was 3D printed and consisted of interlocking top lid and bottom base with inlet and outlet channels. The path length of the spiral device consisted of 20 microchannels with 6.0-feet length. Device accommodated 28 gm of non-hemolytic G-EpCAM compositions. CFD analysis showed 3.8 mm as the ideal channel diameter and 2mm as the superlative G-EpCAM diameter for maximal cells and CTC capture with minimal blood hemolysis (less than 1%) as compared to control. Series 1 and 2 device indicated 90% and 85% cell capture efficiency, respectively using G-EpCAM devices indicating highest interactions and efficiency with cells. Conversely, the first device in series captured the highest cells. In addition, the efficiency improved as the number of G-EpCAM compositions was increased. We accounted device to capture CTCs with specificity having G-EpCAM composition and observed no hemolysis and non-specific interactions with other blood cells like RBCs or leukocytes. Conclusions: Continuous CTC removal from cancer patient’s blood circulation using such device offers promising therapeutic utility in stemming aggressive metastatic invasion and progression for improving the overall survival of epithelial origin cancer patients. Clinical trial information: CTRI U1111/1192-3951.

PH-Responsive Carbon Nanotube Film-Based Microfluidic Chip for Efficient Capture and Release of Cancer Cells

pH-Responsive Carbon Nanotube Film-Based Microfluidic Chip for Efficient Capture and Release of Cancer Cells

A Transparent Semiconducting Surface for Capturing and Releasing Single Cells from a Complex Cell Mixture.

Selective isolation of individual target cells from a heterogeneous population is technically challenging; however, the ability to retrieve single cells can have high significance in various aspects of biological research. Here, we present a new photoelectrochemical surface based on a transparent electrode that is compatible with high-resolution fluorescence microscopy for isolating individual rare cells from complex biological samples. This is underpinned by two important factors: (i) careful design of the electrode by patterning discrete Au disks of micron dimension on amorphous silicon-indium tin oxide films and (ii) orthogonal surface chemistry, which modifies the patterned electrode with self-assembly layers of different functionalities, to selectively capture target cells on the Au disks and resist cell binding to the amorphous silicon surface. The co-stimulation of the surface using light from a microscope and an electric potential triggers the reductive desorption of the alkanethiol monolayer from the Au disks to release the single cells of interest from the illuminated regions only. Using circulating tumor cells as a model, we demonstrate the capture of cancer cells on an antibody-coated surface and selective release of single cancer cells with low expression of epithelial cell adhesion molecules.

A Novel Electrochemically Switchable Conductive Polymer Interface for Controlled Capture and Release of Chemical and Biological Entities

AbstractMaterials platforms that enable controlled isolation and subsequent release of chemical/biological entities are in great demand for a diverse range of practical applications. Current technologies lack good control and efficiency of the release, which is needed to preserve the captured targets of interest. Here, this need is addressed by providing a versatile, controllable, electrochemical capture/release interface. The interface consists of a highly porous electrospun membrane, electrodeposited with a thiol‐functionalized 3,4‐ethylenedioxythiophene (EDOT) conductive terpolymer, in which the thiol moiety undergoes oxidation/reduction cycles at moderate potentials (+1.0 and −0.8 V, respectively) to enable capture/release. The fast oxidative capture (1 min) and reductive release (2 min) of a model thiol molecule in a highly controllable manner, followed by successful capture/release of an antibody, are demonstrated. Then, femtosecond laser‐patterning is used to fabricate an array of ≈30 µm pores on the electrospun membrane, subsequently coated with the conducting terpolymer, enabling the highly efficient (>90%), fast (20 min) and selective capture of MCF7 cancer cells with 33% release efficiency when polarized at −0.8 V. The released cells show a high level of viability, indicating the capture and release process does not affect cell survival.

Bifunctional biohybrid magnetically propelled microswimmer

Inspired by the intrinsic helical structures of vessels in banana leaves, a novel bifunctional biohybrid magnetic microswimmer with both environment remediation and biomedical application was synthesized by a simple chemical method. Here, spiral vessels isolated from banana leaves were used as biological templates, where nickel layers with magnetic properties and Fe3+-TA (tannic acid, TA) films with pH-dependent degradation capabilities were modified on their surfaces by simple chemical reactions in solution at room temperature. The obtained magnetic helical (Helix/Ni/Fe3+-TA) microswimmers can be efficiently propelled in either pollutant solution (rhodamine B, RhB) or biofluid solution (DMEM culture medium and fetal bovine serum) in the presence of a magnetic field. The developed Helix/Ni/Fe3+-TA microswimmers can be used to efficiently degrade organic pollutant RhB due to the Fe3+-TA films which can produce reactive oxygen species under the action of hydrogen peroxide (H2O2). Moreover, due to the specific chemical interaction between the microswimmers and the surface of cancer cells, the microswimmers can precisely capture cancer cells and then produce heat under light irradiation to kill those cells. These novel helical microswimmers with simple and economic fabrication, efficient magnetic propulsion and diverse functionality show great potential in future environmental and biological applications.