From 3D printing to 3D bioprinting: the material properties of polymeric material and its derived bioink for achieving tissue specific architectures.

The application of 3D printing technologies fields for biological tissues, organs, and cells in the context of medical and biotechnology applications requires a significant amount of innovation in a narrow printability range. 3D bioprinting is one such way of addressing critical design challenges in tissue engineering. In a more general sense, 3D printing has become essential in customized implant designing, faithful reproduction of microenvironmental niches, sustainable development of implants, in the capacity to address issues of effective cellular integration, and long-term stability of the cellular constructs in tissue engineering. This review covers various aspects of 3D bioprinting, describes the current state-of-the-art solutions for all aforementioned critical issues, and includes various illustrative representations of technologies supporting the development of phases of 3D bioprinting. It also demonstrates several bio-inks and their properties crucial for being used for 3D printing applications. The review focus on bringing together different examples and current trends in tissue engineering applications, including bone, cartilage, muscles, neuron, skin, esophagus, trachea, tympanic membrane, cornea, blood vessel, immune system, and tumor models utilizing 3D printing technology and to provide an outlook of the future potentials and barriers.

Poloxamer/Poly(ethylene glycol) Self-Healing Hydrogel for High-Precision Freeform Reversible Embedding of Suspended Hydrogel.

Freeform reversible embedding of suspended hydrogel (FRESH) is an additive manufacturing technique enabling the 3D printing of soft materials with low or no yield stress. The printed material is embedded during the process until its solidification. From the literature, FRESH abilities are self-healing, reusability, suspending, thermal stability, and high-precision printing. This study proposes a new support hydrogel bath formulation for FRESH 3D printing. To do so, a poloxamer micellar thermoreversible hydrogel is tuned through the addition of poly(ethylene glycol) (PEG) to adapt rheological properties. PEG macromolecules interact with poly(ethylene oxide) blocks of poloxamer and favor micelle dehydration, and then decreasing the gelation temperature, the yield stress, and the viscosity. Parameters such as the Oldroyd number and the Rayleigh-Plateau instability, both dependent on yield stress, were studied to determine their impact on the FRESH 3D printing resolution and accuracy. It was found that print accuracy of embedded parts increases with increasing yield stress but then the self-healing property gets limited, leading to crevasse formation. The usefulness of this approach is distinctly demonstrated through a six-axis printing of a highly complex silicone anatomical model. Printing fidelity of 96.0 ± 3.58% (5-40 mm printed parts) is thus achieved using the newly formulated FRESH material, while only 56.0 ± 0.76% fidelity is obtained using the standard formulation. The present study thus showed that complex FRESH 3D printing of soft materials is possible in this tunable hydrogel and that parts can be manufactured on an industrial scale, thanks to the reusability of the support bath.

Integrated graphene quantum dot decorated functionalized nanosheet biosensor for mycotoxin detection.

Decoration of graphene quantum dots (GQDs) on molybdenum disulfide (MoS2) nanosheets serves as an active electrode material which enhances the electrochemical performance of the analyte detection system. Herein, ionic surfactant cetyltrimethylammonium bromide (CTAB)-exfoliated MoS2 nanosheets decorated with GQD material are used to construct an electrochemical biosensor for aflatoxin B1 (AFB1) detection. An antibody of AFB1 (aAFB1) was immobilized on the electrophoretically deposited MoS2@GQDs film on the indium tin oxide (ITO)-coated glass surface using a crosslinker for the fabrication of the biosensor. The immunosensing study investigated by the electrochemical method revealed a signal response in the range of 0.1 to 3.0 ng/mL AFB1 concentration with a detection limit of 0.09 ng/mL. Also, electrochemical parameters such as diffusion coefficient and heterogeneous electron transfer (HET) were calculated and found to be 1.67 × 10-5 cm2/s and 2 × 10-5 cm/s, respectively. The effective conjugation of MoS2@GQDs that provides abundant exposed edge sites, large surface area, improved electrical conductivity, and electrocatalytic activity has led to an excellent biosensing performance with enhanced electrochemical parameters. Validation of the fabricated immunosensor was performed in a spiked maize sample, and a good percentage of recoveries within an acceptable range were obtained (80.2 to 98.3%).Graphical abstract.

A new functional membrane protein microarray based on tethered phospholipid bilayers.

A new prototype of a membrane protein biochip is presented in this article. This biochip was created by the combination of novel technologies of peptide-tethered bilayer lipid membrane (pep-tBLM) formation and solid support micropatterning. Pep-tBLMs integrating a membrane protein were obtained in the form of microarrays on a gold chip. The formation of the microspots was visualized in real-time by surface plasmon resonance imaging (SPRi) and the functionality of a GPCR (CXCR4), reinserted locally into microwells, was assessed by ligand binding studies. In brief, to achieve micropatterning, P19-4H, a 4 histidine-possessing peptide spacer, was spotted inside microwells obtained on polystyrene-coated gold, and Ni-chelating proteoliposomes were injected into the reaction chamber. Proteoliposome binding to the peptide was based on metal-chelate interaction. The peptide-tethered lipid bilayer was finally obtained by addition of a fusogenic peptide (AH peptide) to promote proteoliposome fusion. The CXCR4 pep-tBLM microarray was characterized by surface plasmon resonance imaging (SPRi) throughout the building-up process. This new generation of membrane protein biochip represents a promising method of developing a screening tool for drug discovery.

Adding Biomolecular Recognition Capability to 3D Printed Objects.

Three-dimensional (3D) printing technologies will impact the biosensor community in the near future, at both the sensor prototyping level and the sensing layer organization level. The present study aimed at demonstrating the capacity of one 3D printing technique, digital light processing (DLP), to produce hydrogel sensing layers with 3D shapes that are unattainable using conventional molding procedures. The first model of the sensing layer was composed of a sequential enzymatic reaction (glucose oxidase and peroxidase), which generated a chemiluminescent signal in the presence of glucose and luminol. Highly complex objects with assembly properties (fanciful ball, puzzle pieces, 3D pixels, propellers, fluidic and multicompartments) with mono-, di-, and tricomponents configurations were achieved, and the activity of the entrapped enzymes was demonstrated. The second model was a sandwich immunoassay protocol for the detection of brain natriuretic peptide. Here, highly complex propeller shape sensing layers were produced, and the recognition capability of the antibodies was elucidated. The present study opens then the path to a totally new field of development of multiplex sensing layers, printed separately and assembled on demand to create complex sensing systems.

Protein-peptide arrays for detection of specific anti-hepatitis D virus (HDV) genotype 1, 6, and 8 antibodies among HDV-infected patients by surface plasmon resonance imaging.

Liver diseases linked to hepatitis B-hepatitis D virus co- or superinfections are more severe than those during hepatitis B virus (HBV) monoinfection. The diagnosis of hepatitis D virus (HDV) infection therefore remains crucial in monitoring patients but is often overlooked. To integrate HDV markers into high-throughput viral hepatitis diagnostics, we studied the binding of anti-HDV antibodies (Abs) using surface plasmon resonance imaging (SPRi). We focused on the ubiquitous HDV genotype 1 (HDV1) and the more uncommon African-HDV6 and HDV8 genotypes to define an array with recombinant proteins or peptides. Full-length and truncated small hepatitis D antigen (S-HDAg) recombinant proteins of HDV genotype 1 (HDV1) and 11 HDV peptides of HDV1, 6, and 8, representing various portions of the delta antigen were grafted onto biochips, allowing SPRi measurements to be made. Sixteen to 17 serum samples from patients infected with different HDV genotypes were injected onto protein and peptide chips. In all, Abs against HDV proteins and/or peptides were detected in 16 out of 17 infected patients (94.12%), although the amplitude of the SPR signal varied. The amino-terminal part of the protein was poorly immunogenic, while epitope 65-80, exposed on the viral ribonucleoprotein, may be immunodominant, as 9 patient samples led to a specific SPR signal on peptide 65 type 1 (65#1), independently of the infecting genotype. In this pilot study, we confirmed that HDV infection screening based on the reactivity of patient Abs against carefully chosen HDV peptides and/or proteins can be included in a syndrome-based viral hepatitis diagnostic assay. The preliminary results indicated that SPRi studying direct physical HDAg-anti-HDV Ab interactions was more convenient using linear peptide epitopes than full-length S-HDAg proteins, due to the regeneration process, and may represent an innovative approach for a hepatitis syndrome-viral etiology-exploring array.

Disease map-based biomarker selection and pre-validation for bladder cancer diagnostic.

CONTEXT: Urinary biomarkers are promising as simple alternatives to cystoscopy for the diagnosis of de novo and recurrent bladder cancer. OBJECTIVE: To identify a highly sensitive and specific biomarker candidate set with potential clinical utility in bladder cancer. MATERIALS AND METHODS: Urinary biomarker concentrations were determined by ELISA. The performance of individual markers and marker combinations was assessed using ROC analysis. RESULTS: A five-biomarker panel (IL8, MMP9, VEGFA, PTGS2 and EN2) was defined from the candidate set. DISCUSSION AND CONCLUSION: This panel showed a better overall performance than the best individual marker. Further validation studies are needed to evaluate its clinical utility in bladder cancer.

Tuning viscoelasticity and stiffness in bioprinted hydrogels for enhanced 3D cell culture: A multi-scale mechanical analysis.

Bioprinted hydrogels are extensively studied to provide an artificial matrix for 3D cell culture. The success of bioprinting hydrogels relies on fine-tuning their rheology and composition to achieve shear-thinning behavior. However, a challenge arises from the limited viscoelastic and stiffness range accessible from a single hydrogel formulation. Nevertheless, hydrogel mechanical properties are recognized as essential cues influencing cell phenotype, migration, and differentiation. Thus, it is crucial to develop a system to easily modulate bioprinted hydrogels' mechanical behaviors. In this work, we modulated the viscoelastic properties and stiffness of bioprinted hydrogels composed of fibrinogen, alginate, and gelatin by tuning the crosslinking bath solution. Various concentrations of calcium ionically crosslinked alginate, while transglutaminase crosslinked gelatin. Subsequently, we characterized the mechanical behavior of our bioprinted hydrogels from the nanoscale to the macroscale. This approach enabled the production of diverse bioprinted constructs, either with similar elastic behavior but different elastic moduli or with similar elastic moduli but different viscoelastic behavior from the same hydrogel formulation. Culturing fibroblasts in the hydrogels for 33 days revealed a preference for cell growth and matrix secretion in the viscoelastic hydrogels. This work demonstrates the suitability of the method to decouple the effects of material mechanical from biochemical composition cues on 3D cultured cells.

Three-Dimensionally Printed Biphasic Calcium Phosphate Ceramic Substrates as the Sole Inducer of Osteogenic Differentiation in Stromal Vascular Fraction Cells.

The stromal vascular fraction (SVF) is a derivate of fat tissue comprising both adipose-derived mesenchymal stem cells and endothelial cells and serves as a promising cell source for engineering vascularized bone tissues. Its combination with osteoconductive biphasic calcium phosphate (BCP) ceramic may represent a point-of-care agent for bone reconstruction. Here we assessed the proliferation and osteogenic differentiation capacities of SVF on 3D printed BCP implants, in comparison with isolated adipose-derived mesenchymal stem cells (AD-MSCs). AD-MSCs and SVF isolated from human donors were seeded on plastic or 3D printed BCP ceramics with sinusoidal or gyroid macrotopography and cultured in the presence or absence of osteogenic factors. Vascular, hematopoietic and MSC surface markers were assessed by flow cytometry whereas osteogenic activity was investigated through alizarin red staining and alkaline phosphatase activity. Osteogenic factors were necessary to trigger osteogenic activity when cells were cultured on plastic, without significant difference observed between the two cell populations. Interestingly, osteogenic activity was observed on BCP implants in the absence of differentiation factors, without significant difference in level activity between the two cell populations and macrotopography. This study offers supportive data for the use of combined BCP scaffolds with SVF in a perspective of a one-step surgical procedure for bone regeneration.

Confined bioprinting and culture in inflatable bioreactor for the sterile bioproduction of tissues and organs.

The future of organ and tissue biofabrication strongly relies on 3D bioprinting technologies. However, maintaining sterility remains a critical issue regardless of the technology used. This challenge becomes even more pronounced when the volume of bioprinted objects approaches organ dimensions. Here, we introduce a novel device called the Flexible Unique Generator Unit (FUGU), which is a unique combination of flexible silicone membranes and solid components made of stainless steel. Alternatively, the solid components can also be made of 3D printed medical-grade polycarbonate. The FUGU is designed to support micro-extrusion needle insertion and removal, internal volume adjustment, and fluid management. The FUGU was assessed in various environments, ranging from custom-built basic cartesian to sophisticated 6-axis robotic arm bioprinters, demonstrating its compatibility, flexibility, and universality across different bioprinting platforms. Sterility assays conducted under various infection scenarios highlight the FUGU's ability to physically protect the internal volume against contaminations, thereby ensuring the integrity of the bioprinted constructs. The FUGU also enabled bioprinting and cultivation of a 14.5 cm3 human colorectal cancer tissue model within a completely confined and sterile environment, while allowing for the exchange of gases with the external environment. This FUGU system represents a significant advancement in 3D bioprinting and biofabrication, paving the path toward the sterile production of implantable tissues and organs.

3D MODEL of an anatomically inert human hand: feasibility study.

OBJECTIVES: Surgery for congenital malformation of the hand is complex and protocols are not available. Simulation could help optimize results. The objective of the present study was to design, produce and assess a 3D-printed anatomical support, to improve success in rare and complex surgeries of the hand. MATERIAL AND METHODS: We acquired MRI imaging of the right hand of a 30 year-old subject, then analyzed and split the various skin layers for segmentation. Thus we created the prototype of a healthy hand, using 3D multi-material and silicone printing devices, and drew up a printing protocol suitable for all patients. We printed a base comprising bones, muscles and tendons, with a multi-material 3D printer, then used a 3D silicone printer for skin and subcutaneous fatty cell tissues in a glove-like shape. To evaluate the characteristics of the prototype, we performed a series of dissections on the synthetic hand and on a cadaveric hand in the anatomy lab, comparing realism, ease of handling and the final result of the two supports, and evaluated their respective advantages in surgical and training contexts. A grading form was given to each surgeon to establish a global score. RESULTS: This evaluation highlighted the positive and negative features of the model. The model avoided intrinsic problems of cadavers, such as muscle rigidity or tissue fragility and atrophy, and enables the anatomy of a specific patient to be rigorously respected. On the other hand, vascular and nervous networks, with their potential anatomical variants, are lacking. This preliminary phase highlighted the advantages and inconveniences of the prototype, to optimize the design and printing of future models. It is an indispensable prerequisite before performing studies in eligible pediatric patients with congenital hand malformation. CONCLUSION: The validation of 3D-printed anatomical model of a human hand opens a large field of applications in the area of preoperative surgical planning. The postoperative esthetic and functional benefit of such pre-intervention supports in complex surgery needs assessing.

3D bioprinted CRC model brings to light the replication necessity of an oncolytic vaccinia virus encoding FCU1 gene to exert an efficient anti-tumoral activity.

The oncolytic virus represents a promising therapeutic strategy involving the targeted replication of viruses to eliminate cancer cells, while preserving healthy ones. Despite ongoing clinical trials, this approach encounters significant challenges. This study delves into the interaction between an oncolytic virus and extracellular matrix mimics (ECM mimics). A three-dimensional colorectal cancer model, enriched with ECM mimics through bioprinting, was subjected to infection by an oncolytic virus derived from the vaccinia virus (oVV). The investigation revealed prolonged expression and sustained oVV production. However, the absence of a significant antitumor effect suggested that the virus's progression toward non-infected tumoral clusters was hindered by the ECM mimics. Effective elimination of tumoral cells was achieved by introducing an oVV expressing FCU1 (an enzyme converting the prodrug 5-FC into the chemotherapeutic compound 5-FU) alongside 5-FC. Notably, this efficacy was absent when using a non-replicative vaccinia virus expressing FCU1. Our findings underscore then the crucial role of oVV proliferation in a complex ECM mimics. Its proliferation facilitates payload expression and generates a bystander effect to eradicate tumors. Additionally, this study emphasizes the utility of 3D bioprinting for assessing ECM mimics impact on oVV and demonstrates how enhancing oVV capabilities allows overcoming these barriers. This showcases the potential of 3D bioprinting technology in designing purpose-fit models for such investigations.

Challenges in Nasal Cartilage Tissue Engineering to Restore the Shape and Function of the Nose.

The repair of nasal septal cartilage is a key challenge in cosmetic and functional surgery of the nose, as it determines its shape and its respiratory function. Supporting the dorsum of the nose is essential for both the prevention of nasal obstruction and the restoration of the nose structure. Most surgical procedures to repair or modify the nasal septum focus on restoring the external aspect of the nose by placing a graft under the skin, without considering respiratory concerns. Tissue engineering offers a more satisfactory approach, in which both the structural and biological roles of the nose are restored. To achieve this goal, nasal cartilage engineering research has led to the development of scaffolds capable of accommodating cartilaginous extracellular matrix-producing cells, possessing mechanical properties close to those of the nasal septum, and retaining their structure after implantation in vivo. The combination of a non-resorbable core structure with suitable mechanical properties and a biocompatible hydrogel loaded with autologous chondrocytes or mesenchymal stem cells is a promising strategy. However, the stability and immunotolerance of these implants are crucial parameters to be monitored over the long term after in vivo implantation, to definitively assess the success of nasal cartilage tissue engineering. Here, we review the tissue engineering methods to repair nasal cartilage, focusing on the type and mechanical characteristics of the biomaterials; cell and implantation strategy; and the outcome with regard to cartilage repair.

Targeting the Complexity of In Vitro Skin Models: A Review of Cutting-Edge Developments.

Skin in vitro models offer much promise for research, testing drugs, cosmetics, and medical devices, reducing animal testing and extensive clinical trials. There are several in vitro approaches to mimicking human skin behavior, ranging from simple cell monolayer to complex organotypic and bioengineered 3-dimensional models. Some have been approved for preclinical studies in cosmetics, pharmaceuticals, and chemicals. However, development of physiologically reliable in vitro human skin models remains in its infancy. This review reports on advances in in vitro complex skin models to study skin homeostasis, aging, and skin disease.

Space radiation damage rescued by inhibition of key spaceflight associated miRNAs.

Our previous research revealed a key microRNA signature that is associated with spaceflight that can be used as a biomarker and to develop countermeasure treatments to mitigate the damage caused by space radiation. Here, we expand on this work to determine the biological factors rescued by the countermeasure treatment. We performed RNA-sequencing and transcriptomic analysis on 3D microvessel cell cultures exposed to simulated deep space radiation (0.5 Gy of Galactic Cosmic Radiation) with and without the antagonists to three microRNAs: miR-16-5p, miR-125b-5p, and let-7a-5p (i.e., antagomirs). Significant reduction of inflammation and DNA double strand breaks (DSBs) activity and rescue of mitochondria functions are observed after antagomir treatment. Using data from astronaut participants in the NASA Twin Study, Inspiration4, and JAXA missions, we reveal the genes and pathways implicated in the action of these antagomirs are altered in humans. Our findings indicate a countermeasure strategy that can potentially be utilized by astronauts in spaceflight missions to mitigate space radiation damage.

Carbon microarrays for the direct impedimetric detection of Bacillus anthracis using Gamma phages as probes.

A direct and efficient impedimetric method is presented for the detection of Bacillus anthracis Sterne vegetative cells, using Gamma phages as probes attached to screen-printed carbon electrode microarrays. The carbon electrodes were initially functionalized through cyclic-voltammetric reduction of a nitro-aryl diazonium moiety, followed by further reduction of nitro groups to amino groups, and finally by treatment with glutaraldehyde. Functionalization (probe immobilization) using Gamma phages was verified by XPS and TOF-SIM experiments. The Gamma phage-modified microarrays were then used to detect B. anthracis Sterne bacteria in aqueous electrolyte media. Faradaic impedimetric detection of bacteria in KCl solution containing the ferri/ferro cyanide redox couple shows a gradual increase in Z' (real impedance) values, taken from the extrapolation of the linear portion of Nyquist plots in the low frequency range, for sensors placed in contact with increasing concentrations of B. anthracis. ΔZ' values vary from 700 to 5300 Ohms for bacteria concentrations ranging from 10(2) to 10(8) cfu mL(-1). These shifts in Z' are attributed to a decrease in diffusion controlled charge transfer to the electrode surface following capture of intact B. anthracis. No significant ΔZ' was observed for control experiments using E. coli. K12 as a non-specific target, even at a concentration of 10(8) cfu mL(-1).

In situ transient transfection of 3D cell cultures and tissues, a promising tool for tissue engineering and gene therapy.

Various research fields use the transfection of mammalian cells with genetic material to induce the expression of a target transgene or gene silencing. It is a tool widely used in biological research, bioproduction, and therapy. Current transfection protocols are usually performed on 2D adherent cells or suspension cultures. The important rise of new gene therapies and regenerative medicine in the last decade raises the need for new tools to empower the in situ transfection of tissues and 3D cell cultures. This review will present novel in situ transfection methods based on a chemical or physical non-viral transfection of cells in tissues and 3D cultures, discuss the advantages and remaining gaps, and propose future developments and applications.

Increasing Collagen to Bioink Drives Mesenchymal Stromal Cells-Chondrogenesis from Hyaline to Calcified Layers.

The bioextrusion of mesenchymal stromal cells (MSCs) directly seeded in a bioink enables the production of three-dimensional (3D) constructs, promoting their chondrogenic differentiation. Our study aimed to evaluate the effect of different type I collagen concentrations in the bioink on MSCs' chondrogenic differentiation. We printed 3D constructs using an alginate, gelatin, and fibrinogen-based bioink cellularized with MSCs, with four different quantities of type I collagen addition (0.0, 0.5, 1.0, and 5.0 mg per bioink syringe). We assessed the influence of the bioprinting process, the bioink composition, and the growth factor (TGF-ꞵ1) on the MSCs' survival rate. We confirmed the biocompatibility of the process and the bioinks' cytocompatibility. We evaluated the chondrogenic effects of TGF-ꞵ1 and collagen addition on the MSCs' chondrogenic properties through macroscopic observation, shrinking ratio, reverse transcription polymerase chain reaction, glycosaminoglycan synthesis, histology, and type II collagen immunohistochemistry. The bioink containing 0.5 mg of collagen produces the richest hyaline-like extracellular matrix, presenting itself as a promising tool to recreate the superficial layer of hyaline cartilage. The bioink containing 5.0 mg of collagen enhances the synthesis of a calcified matrix, making it a good candidate for mimicking the calcified cartilaginous layer. Type I collagen thus allows the dose-dependent design of specific hyaline cartilage layers.

3D Bioprinting in Microgravity: Opportunities, Challenges, and Possible Applications in Space.

3D bioprinting has developed tremendously in the last couple of years and enables the fabrication of simple, as well as complex, tissue models. The international space agencies have recognized the unique opportunities of these technologies for manufacturing cell and tissue models for basic research in space, in particular for investigating the effects of microgravity and cosmic radiation on different types of human tissues. In addition, bioprinting is capable of producing clinically applicable tissue grafts, and its implementation in space therefore can support the autonomous medical treatment options for astronauts in future long term and far-distant space missions. The article discusses opportunities but also challenges of operating different types of bioprinters under space conditions, mainly in microgravity. While some process steps, most of which involving the handling of liquids, are challenging under microgravity, this environment can help overcome problems such as cell sedimentation in low viscous bioinks. Hopefully, this publication will motivate more researchers to engage in the topic, with publicly available bioprinting opportunities becoming available at the International Space Station (ISS) in the imminent future.

High-throughput multiplexed competitive immunoassay for pollutants sensing in water.

The present study described the development and evaluation of a new fully automated multiplex competitive immunoassay enabling the simultaneous detection of five water pollutants (okadaic acid (OA), 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine (atrazine), 2.4-dichlorophenoxyacetic acid (2,4-D), 2,4,6-trinitrotoluene (TNT), and 1,3,5-trinitroperhydro-1,3,5-triazine (RDX)). The technology is taking advantage of an optical-clear pressure-sensitive adhesive on which biomolecules can be immobilized and that can be integrated within a classical 96-well format. The optimization of the microarray composition and cross-reaction was performed using an original approach where probe molecules (haptens) were conjugated to different carriers such as protein (bovine serum albumin or ovalbumin), amino-functionalized latex beads, or dextran polymer and arrayed at the surface of the adhesive. A total of 17 different probes were then arrayed together with controls on the adhesive surface and screened toward their specific reactivity and cross-reactivity. Once optimized, the complete setup was used for the detection of the five target molecules (less than 3 h for 96 samples). Limits of detection of 0.02, 0.01, 0.01, 100, and 0.02 µg L(-1) were found for OA, atrazine, 2,4-D, TNT, and RDX, respectively. The proof of concept of the multiplex competitive detection (semiquantitative or qualitative) of the five pollutants was also demonstrated on 16 spiked samples.