ABSTRACT
Photon-upconversion nanoparticles (UCNP) have already been established as labels for affinity assays in analog and digital formats. Here, advanced, or smart, systems based on UCNPs coated with active shells, fluorescent dyes, and metal and semiconductor nanoparticles participating in energy transfer reactions are reviewed. In addition, switching elements can be embedded in such assemblies and provide temporal and spatial control of action, which is important for intracellular imaging and monitoring activities. Demonstration and critical comments on representative approaches demonstrating the progress in the use of such UCNPs in bioanalytical assays, imaging, and monitoring of target molecules in cells are reported, including particular examples in the field of cancer theranostics.
Subject(s)
Nanoparticles , Photons , Humans , Nanoparticles/chemistry , Fluorescent Dyes/chemistry , Animals , Neoplasms/diagnostic imaging , Neoplasms/diagnosis , Optical ImagingABSTRACT
Early-stage diagnosis of prostatic carcinoma is essential for successful treatment and, thus, significant prognosis improvement. In laboratory practice, the standard non-invasive diagnostic approach is the immunochemical detection of the associated biomarker, prostate-specific antigen (PSA). Ultrasensitive detection of PSA is essential for both diagnostic and recurrence monitoring purposes. To achieve exceptional sensitivity, we have developed a microfluidic device with a flow-through cell for single-molecule analysis using photon-upconversion nanoparticles (UCNPs) as a detection label. For this purpose, magnetic microparticles (MBs) were first optimized for the capture and preconcentration of PSA and then used to implement a bead-based upconversion-linked immunoassay (ULISA) in the microfluidic device. The digital readout based on counting single nanoparticle-labeled PSA molecules on MBs enabled a detection limit of 1.04 pg mL-1 (36 fM) in 50% fetal bovine serum, which is an 11-fold improvement over the respective analog MB-based ULISA. The microfluidic technique conferred several other advantages, such as easy implementation and the potential for achieving high-throughput analysis. Finally, it was proven that the microfluidic setup is suitable for clinical sample analysis, showing a good correlation with a reference electrochemiluminescence assay (recovery rates between 97% and 105%).
Subject(s)
Prostate-Specific Antigen , Prostate-Specific Antigen/analysis , Prostate-Specific Antigen/blood , Humans , Microfluidic Analytical Techniques/instrumentation , Male , Nanoparticles/chemistry , Immunoassay/instrumentation , Immunoassay/methods , Limit of Detection , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/bloodABSTRACT
Dot-blot immunoassays are widely used for the user-friendly detection of clinical biomarkers. However, the majority of dot-blot assays have only limited sensitivity and are only used for qualitative or semiquantitative analysis. To overcome this limitation, we have employed labels based on photon-upconversion nanoparticles (UCNPs) that exhibit anti-Stokes luminescence and can be detected without optical background interference. First, the dot-blot immunoassay on a nitrocellulose membrane was optimized for the quantitative analysis of human serum albumin (HSA), resulting in a limit of detection (LOD) of 0.19 ng/mL and a signal-to-background ratio (S/B) of 722. Commercial quantum dots were used as a reference label, reaching the LOD of 4.32 ng/mL and the S/B of 3, clearly indicating the advantages of UCNPs. In addition, the potential of UCNP-based dot-blot for real sample analysis was confirmed by analyzing spiked urine samples, reaching the LOD of 0.24 ng/mL and recovery rates from 79 to 123%. Furthermore, we demonstrated the versatility and robustness of the assay by adapting it to the detection of two other clinically relevant biomarkers, prostate-specific antigen (PSA) and cardiac troponin (cTn), reaching the LODs in spiked serum of 9.4 pg/mL and 0.62 ng/mL for PSA and cTn, respectively. Finally, clinical samples of patients examined for prostate cancer were analyzed, achieving a strong correlation with the reference electrochemiluminescence immunoassay (recovery rates from 89 to 117%). The achieved results demonstrate that UCNPs are highly sensitive labels that enable the development of dot-blot immunoassays for quantitative analysis of low-abundance biomarkers.
Subject(s)
Biomarkers , Limit of Detection , Nanoparticles , Prostate-Specific Antigen , Humans , Immunoassay/methods , Nanoparticles/chemistry , Prostate-Specific Antigen/blood , Prostate-Specific Antigen/analysis , Biomarkers/blood , Biomarkers/urine , Biomarkers/analysis , Quantum Dots/chemistry , Serum Albumin, Human/analysis , Serum Albumin, Human/urine , MaleABSTRACT
BACKGROUND: Laser-induced breakdown spectroscopy (LIBS) is a well-recognized analytical technique used for elemental analysis. This method is gaining considerable attention also in biological applications thanks to its ability for spatial mapping and elemental imaging. The implementation of LIBS in the biomedical field is based on the detection of metals or other elements that either naturally occur in the samples or are present artificially. The artificial implementation of nanoparticle labels (Tag-LIBS) enables the use of LIBS as a readout technique for immunochemical assays. However, one of the biggest challenges for LIBS to meet immunoassay readout standards is its sensitivity. RESULTS: This paper focuses on the improvement of LIBS sensitivity for the readout of nanoparticle-based immunoassays. First, the LIBS setup was optimized on photon-upconversion nanoparticle (UCNP) droplets deposited on the microtiter plate wells. Two collection optics systems were compared, with single pulse (SP) and collinear double pulse (DP) LIBS arrangements. By deploying the second laser pulse, the sensitivity was improved up to 30 times. The optimized SP and DP setups were then employed for the indirect detection of human serum albumin based on immunoassay with UCNP-based labels. Compared to our previous LIBS study, the detection limit was enhanced by two orders of magnitude, from 10 ng mL-1 to 0.29 ng mL-1. In addition, two other immunochemical methods were used for reference, based on the readout of upconversion luminescence of UCNPs and absorbance measurement with enzyme labels. Finally, the selectivity of the assay was tested and the practical potential of Tag-LIBS was demonstrated by the successful analysis of urine samples. SIGNIFICANCE AND NOVELTY: In this work, we improved the sensitivity of the Tag-LIBS method by combining new labels based on UCNPs with the improved collection optics and collinear DP configuration. In the instrumental setup optimization, the DP LIBS showed better sensitivity and signal-to-noise ratio than SP. The optimizations allowed the LIBS readout to surpass the sensitivity of enzyme immunoassay, approaching the qualities of upconversion luminescence readout, which is nowadays a state-of-the-art readout technique.
Subject(s)
Nanoparticles , Humans , Spectrum Analysis/methods , Nanoparticles/chemistry , Immunoassay/methods , Lasers , MetalsABSTRACT
The three decades of experience with piezoelectric devices applied in the field of bioanalytical chemistry are shared. After introduction to principles and suitable measuring approaches, active and passive methods based on oscillators and impedance analysis, respectively, the focus is directed towards biosensing approaches. Immunosensing examples are provided, followed by other affinity sensing approaches based on hybridization of nucleic acids, aptamers, monitoring of enzyme activities, and detection of pathogenic microbes. The combination of piezosensors with cell lines and testing of drugs is highlighted, including mechanically active cells. The combination of piezosensors with other measuring techniques providing original hybrid devices is briefly discussed.
Subject(s)
Nucleic Acids , Cell Line , Electric Impedance , OligonucleotidesABSTRACT
Interactions between living cells and nanoparticles are extensively studied to enhance the delivery of therapeutics. Nanoparticles size, shape, stiffness, and surface charge are regarded as the main features able to control the fate of cell-nanoparticle interactions. However, the clinical translation of nanotherapies has so far been limited, and there is a need to better understand the biology of cell-nanoparticle interactions. This study investigates the role of cellular mechanosensitive components in cell-nanoparticle interactions. It is demonstrated that the genetic and pharmacologic inhibition of yes-associated protein (YAP), a key component of cancer cell mechanosensing apparatus and Hippo pathway effector, improves nanoparticle internalization in triple-negative breast cancer cells regardless of nanoparticle properties or substrate characteristics. This process occurs through YAP-dependent regulation of endocytic pathways, cell mechanics, and membrane organization. Hence, the study proposes targeting YAP may sensitize triple-negative breast cancer cells to chemotherapy and increase the selectivity of nanotherapy.
Subject(s)
Nanoparticles , Triple Negative Breast Neoplasms , Humans , Signal Transduction/physiology , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/metabolism , YAP-Signaling ProteinsABSTRACT
Advances in the development of new biorecognition elements, nanoparticle-based labels as well as instrumentation have inspired the design of new bioaffinity assays. This review critically discusses the potential of nanoparticles to replace current enzymatic or molecular labels in immunoassays and other bioaffinity assays. Successful implementations of nanoparticles in commercial assays and the need for rapid tests incorporating nanoparticles in different roles such as capture support, signal generation elements, and signal amplification systems are highlighted. The limited number of nanoparticles applied in current commercial assays can be explained by challenges associated with the analysis of real samples (e.g., blood, urine, or nasal swabs) that are difficult to resolve, particularly if the same performance can be achieved more easily by conventional labels. Lateral flow assays that are based on the visual detection of the red-colored line formed by colloidal gold are a notable exception, exemplified by SARS-CoV-2 rapid antigen tests that have moved from initial laboratory testing to widespread market adaption in less than two years.
Subject(s)
Nanoparticles , Immunoassay , Sensitivity and SpecificityABSTRACT
An electrochemical impedimetric biosensor for human serum albumin (HSA) determination is proposed. The biosensor is based on water-phase assembled nanocomposites made of 2D WS2 nanoflakes and Au nanoparticles (AuNPs). The WS2 has been produced using a liquid-phase exfoliation strategy assisted by sodium cholate, obtaining a water-stable suspension that allowed the straightforward decoration with AuNPs directly in the aqueous phase. The resulting WS2/Au nanocomposite has been characterized by atomic force microscopy and Raman spectroscopy and, then, employed to modify screen-printed electrodes. Good electron-transfer features have been achieved. An electrochemical immunosensing platform has been assembled exploiting cysteamine-glutaraldehyde covalent chemistry for antibody (Ab) immobilization. The resulting immunosensor exhibited good sensitivity for HSA detection (LOD = 2 ng mL-1), with extended linear range (0.005 - 100 µg mL-1), providing a useful analytical tool for HSA determination in urine at relevant clinical ranges for microalbuminuria screening. The HSA quantification in human urine samples resulted in recoveries from 91.8 to 112.4% and was also reproducible (RSD < 7.5%, n = 3), with marked selectivity. This nanocomposite, thanks to the reliable performance and the ease of the assembling strategy, is a promising alternative for electrochemical immunosensing of health relevant markers.
Subject(s)
Biosensing Techniques , Metal Nanoparticles , Nanocomposites , Humans , Metal Nanoparticles/chemistry , Water , Gold/chemistry , Biosensing Techniques/methods , Immunoassay/methods , Serum Albumin, Human , Nanocomposites/chemistryABSTRACT
The COVID-19 crisis requires fast and highly sensitive tests for the early stage detection of the SARS-CoV-2 virus. For detecting the nucleocapsid protein (N protein), the most abundant viral antigen, we have employed upconversion nanoparticles that emit short-wavelength light under near-infrared excitation (976 nm). The anti-Stokes emission avoids autofluorescence and light scattering and thus enables measurements without optical background interference. The sandwich upconversion-linked immunosorbent assay (ULISA) can be operated both in a conventional analog mode and in a digital mode based on counting individual immune complexes. We have investigated how different antibody combinations affect the detection of the wildtype N protein and the detection of SARS-CoV-2 (alpha variant) in lysed culture fluid via the N protein. The ULISA yielded a limit of detection (LOD) of 1.3 pg/mL (27 fM) for N protein detection independent of the analog or digital readout, which is approximately 3 orders of magnitude more sensitive than conventional enzyme-linked immunosorbent assays or commercial lateral flow assays for home testing. In the case of SARS-CoV-2, the digital ULISA additionally improved the LOD by a factor of 10 compared to the analog readout.
Subject(s)
COVID-19 , Immunosorbents , Humans , COVID-19/diagnosis , SARS-CoV-2 , Enzyme-Linked Immunosorbent Assay , Nucleocapsid Proteins , Antibodies, Viral , Sensitivity and SpecificityABSTRACT
Nanoporous surfaces are promising for label-free electrochemical biosensing. We formed nanopores directly on the electrode surface by means of assembling a dense layer of nonconductive nanoparticles. In our model affinity biosensor, covalent attachment of albumin protein on top of 40 nm polystyrene nanoparticles represented a capture of an analyte, resulting in blockage of the nanopores. Different bulk concentrations of the ferro/ferricyanide redox pair were probed by Faradaic electrochemical impedance spectroscopy and fast chronoamperometry. The character of the redox probe permeation towards the electrode surface differed in dependence on its concentration. These data were compared with the theoretical behavior of the free diffusion according to the Cottrell equation. Both the bulk concentration of the redox probe and the timescale of the experiment affected the performance of the electrochemical detection, demonstrating the importance of controlling these parameters in immunosensing applications.
Subject(s)
Biosensing Techniques , Nanopores , Biosensing Techniques/methods , Dielectric Spectroscopy/methods , Electrodes , Oxidation-Reduction , Electrochemical Techniques , Gold/chemistryABSTRACT
A photoelectrochemical biosensor for malate was developed using an indium tin oxide (ITO) layer deposited on a poly(ethylene terephthalate) plastic sheet as a transparent electrode material for the immobilization of malate dehydrogenase together with CdTe quantum dots. Different approaches were compared for the construction of the bioactive layer; the highest response was achieved by depositing malate dehydrogenase together with CdTe nanoparticles and covering it with a Nafion/water (1:1) mixture. The amperometric signal of this biosensor was recorded during irradiation with a near-UV LED in the flow-through mode. The limit of detection was 0.28 mmol/L, which is adequate for analyzing malic acid levels in drinks such as white wines and fruit juices. The results confirm that the cheap ITO layer deposited on the plastic sheet after cutting into rectangular electrodes allows for the economic production of photoelectrochemical (bio)sensors. The combination of NAD+-dependent malate dehydrogenase with quantum dots was also compatible with such an ITO surface.
Subject(s)
Cadmium Compounds , Quantum Dots , Tin Compounds , Malate Dehydrogenase , Malates , TelluriumABSTRACT
Conventional immunochemical methods used in clinical analysis are often not sensitive enough for early-stage diagnosis, resulting in the need for novel assay formats. Here, we provide a detailed comparison of the effect of different labels and solid supports on the performance of heterogeneous immunoassays. When comparing three types of streptavidin-modified labelsâhorseradish peroxidase, carboxyfluorescein, and photon-upconversion nanoparticles (UCNPs)âUCNPs led to the most sensitive and robust detection of the cancer biomarker prostate-specific antigen. Additionally, we compared the immunoassay formats based on conventional microtiter plates and magnetic microbeads (MBs). In both cases, the highest signal-to-background ratios and the lowest limits of detection (LODs) were obtained by using the UCNP labels. The MB-based upconversion-linked immunosorbent assay carried out with a preconcentration step provided the lowest LOD of 0.46 pg/mL in serum. The results demonstrate that the use of UCNPs and MBs can significantly improve the sensitivity and working range of heterogeneous immunoassays for biomarker detection.
Subject(s)
Immunosorbents , Nanoparticles , Male , Humans , Immunoassay/methods , Limit of Detection , Streptavidin , MagneticsABSTRACT
Extracellular HMGB1 protein is known to induce inflammatory responses leading to an inflammatory storm. The outbreak of the Severe Acute Respiratory Syndrome COVID-19 due to the SARS-CoV-2 virus has resulted in a huge health concern worldwide. Recent data revealed that plasma/serum HMGB1 levels of patients suffering from inflammation-mediated disorders-such as COVID-19, cancer, and autoimmune disorders-correlate positively with disease severity and vice versa. A late release of HMGB1 in sepsis suggests the existence of a wide therapeutic window for treating sepsis. Rapid and accurate methods for the detection of HMGB1 levels in plasma/serum are, therefore, of great importance for monitoring the occurrence, treatment success, and survival prediction of patients with inflammation-mediated diseases. In this review, we briefly explain the role of HMGB1 in the cell, and particularly the involvement of extracellular HMGB1 (released from the cells) in inflammation-mediated diseases, with an emphasis on COVID-19. The current assays to measure HMGB1 levels in human plasma-Western blotting, ELISA, EMSA, and a new approach based on electrochemical immunosensors, including some of our preliminary results-are presented and thoroughly discussed.
Subject(s)
COVID-19 , HMGB1 Protein , Sepsis , Biosensing Techniques , COVID-19/blood , COVID-19/diagnosis , HMGB1 Protein/blood , Humans , Immunoassay , Prognosis , SARS-CoV-2ABSTRACT
The detection of cancer biomarkers in histological samples and blood is of paramount importance for clinical diagnosis. Current methods are limited in terms of sensitivity, hindering early detection of disease. We have overcome the shortcomings of currently available staining and fluorescence labeling methods by taking an integrative approach to establish photon-upconversion nanoparticles (UCNP) as a powerful platform for cancer detection. These nanoparticles are readily synthesized in different sizes to yield efficient and tunable short-wavelength light emission under near-infrared excitation, which eliminates optical background interference of the specimen. Here we present a protocol for the synthesis of UCNPs by high-temperature co-precipitation or seed-mediated growth by thermal decomposition, surface modification by silica or poly(ethylene glycol) that renders the particles resistant to nonspecific binding, and the conjugation of streptavidin or antibodies for biological detection. To detect blood-based biomarkers, we present an upconversion-linked immunosorbent assay for the analog and digital detection of the cancer marker prostate-specific antigen. When applied to immunocytochemistry analysis, UCNPs enable the detection of the breast cancer marker human epidermal growth factor receptor 2 with a signal-to-background ratio 50-fold higher than conventional fluorescent labels. UCNP synthesis takes 4.5 d, the preparation of the antibody-silica-UCNP conjugate takes 3 d, the streptavidin-poly(ethylene glycol)-UCNP conjugate takes 2-3 weeks, upconversion-linked immunosorbent assay takes 2-4 d and immunocytochemistry takes 8-10 h. The procedures can be performed after standard laboratory training in nanomaterials research.
Subject(s)
Nanoparticles , Neoplasms , Biomarkers, Tumor , Humans , Immunosorbents , Male , Nanoparticles/chemistry , Neoplasms/diagnosis , Polyethylene Glycols/chemistry , Silicon Dioxide/chemistry , StreptavidinABSTRACT
The growing incidence of multidrug-resistant bacterial strains presents a major challenge in modern medicine. Antibiotic resistance is often exhibited by Staphylococcus aureus, which causes severe infections in human and animal hosts and leads to significant economic losses. Antimicrobial agents with enzymatic activity (enzybiotics) and phage therapy represent promising and effective alternatives to classic antibiotics. However, new tools are needed to study phage-bacteria interactions and bacterial lysis with high resolution and in real-time. Here, we introduce a method for studying the lysis of S. aureus at the single-cell level in real-time using atomic force microscopy (AFM) in liquid. We demonstrate the ability of the method to monitor the effect of the enzyme lysostaphin on S. aureus and the lytic action of the Podoviridae phage P68. AFM allowed the topographic and biomechanical properties of individual bacterial cells to be monitored at high resolution over the course of their lysis, under near-physiological conditions. Changes in the stiffness of S. aureus cells during lysis were studied by analyzing force-distance curves to determine Young's modulus. This allowed observing a progressive decline in cellular stiffness corresponding to the disintegration of the cell envelope. The AFM experiments were complemented by surface plasmon resonance (SPR) experiments that provided information on the kinetics of phage-bacterium binding and the subsequent lytic processes. This approach forms the foundation of an innovative framework for studying the lysis of individual bacteria that may facilitate the further development of phage therapy.
Subject(s)
Bacteriophages , Staphylococcal Infections , Animals , Humans , Microscopy, Atomic Force , Staphylococcus aureus , Surface Plasmon ResonanceABSTRACT
Sensitive immunoassays are required for troponin, a low-abundance cardiac biomarker in blood. In contrast to conventional (analog) assays that measure the integrated signal of thousands of molecules, digital assays are based on counting individual biomarker molecules. Photon-upconversion nanoparticles (UCNP) are an excellent nanomaterial for labeling and detecting single biomarker molecules because their unique anti-Stokes emission avoids optical interference, and single nanoparticles can be reliably distinguished from the background signal. Here, the effect of the surface architecture and size of UCNP labels on the performance of upconversion-linked immunosorbent assays (ULISA) is critically assessed. The size, brightness, and surface architecture of UCNP labels are more important for measuring low troponin concentrations in human plasma than changing from an analog to a digital detection mode. Both detection modes result approximately in the same assay sensitivity, reaching a limit of detection (LOD) of 10 pg mL-1 in plasma, which is in the range of troponin concentrations found in the blood of healthy individuals.
Subject(s)
Nanoparticles , Humans , Immunoassay , Particle Size , Photons , TroponinABSTRACT
Immunohistochemistry (IHC) and immunocytochemistry (ICC) are widely used to identify cancerous cells within tissues and cell cultures. Even though the optical microscopy evaluation is considered the gold standard, the limited range of useful labels and narrow multiplexing capabilities create an imminent need for alternative readout techniques. Laser-induced breakdown spectroscopy (LIBS) enables large-scale multi-elemental analysis of the surface of biological samples, e.g., thin section or cell pellet. It is, therefore, a potential alternative for IHC and ICC readout of various labels or tags (Tag-LIBS approach). Here, we introduce Tag-LIBS as a method for the specific determination of HER2 biomarker. The cell pellets were labeled with streptavidin-conjugated upconversion nanoparticles (UCNP) through a primary anti-HER2 antibody and a biotinylated secondary antibody. The LIBS scanning enabled detecting the characteristic elemental signature of yttrium as a principal constituent of UCNP, thus indirectly providing a reliable way to differentiate between HER2-positive BT-474 cells and HER2-negative MDA-MB-231 cells. The comparison of results with upconversion optical microscopy and luminescence intensity scanning confirmed that LIBS is a promising alternative for the IHC and ICC readout.
Subject(s)
Biomarkers, Tumor/analysis , Nanoparticles/chemistry , Receptor, ErbB-2/analysis , Antibodies, Immobilized/immunology , Biomarkers, Tumor/immunology , Cell Line, Tumor , Feasibility Studies , Fluorides/chemistry , Fluorides/radiation effects , Humans , Immunohistochemistry/methods , Light , Nanoparticles/radiation effects , Receptor, ErbB-2/immunology , Spectrum Analysis/methods , Thulium/chemistry , Thulium/radiation effects , Yttrium/chemistry , Yttrium/radiation effectsABSTRACT
INTRODUCTION: The present study reports on examination of the effects of encapsulating the tyrosine kinase inhibitors (TKIs) vandetanib and lenvatinib into a biomacromolecular ferritin-based delivery system. METHODS: The encapsulation of TKIs was performed via two strategies: i) using an active reversible pH-dependent reassembly of ferritin´s quaternary structure and ii) passive loading of hydrophobic TKIs through the hydrophobic channels at the junctions of ferritin subunits. After encapsulation, ferritins were surface-functionalized with folic acid promoting active-targeting capabilities. RESULTS: The physico-chemical and nanomechanical analyses revealed that despite the comparable encapsulation efficiencies of both protocols, the active loading affects stability and rigidity of ferritins, plausibly due to their imperfect reassembly. Biological experiments with hormone-responsive breast cancer cells (T47-D and MCF-7) confirmed the cytotoxicity of encapsulated and folate-targeted TKIs to folate-receptor positive cancer cells, but only limited cytotoxic effects to healthy breast epithelium. Importantly, the long-term cytotoxic experiments revealed that compared to the pH-dependent encapsulation, the passively-loaded TKIs exert markedly higher anticancer activity, most likely due to undesired influence of harsh acidic environment used for the pH-dependent encapsulation on the TKIs' structural and functional properties. CONCLUSION: Since the passive loading does not require a reassembly step for which acids are needed, the presented investigation serves as a solid basis for future studies focused on encapsulation of small hydrophobic molecules.
Subject(s)
Drug Delivery Systems , Ferritins/chemistry , Folic Acid/chemistry , Phenylurea Compounds/pharmacology , Piperidines/pharmacology , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , Quinolines/pharmacology , Animals , Antineoplastic Agents/pharmacology , Biocompatible Materials/chemistry , Cell Death/drug effects , Cell Line , Cell Movement/drug effects , Clone Cells , Diffusion , Drug Carriers/chemistry , Horses , Humans , Hydrogen-Ion Concentration , Phenylurea Compounds/chemistry , Piperidines/chemistry , Quinazolines/chemistry , Quinolines/chemistry , Surface PropertiesABSTRACT
Numerous protocols of cardiac differentiation have been established by essentially focusing on specific growth factors on human pluripotent stem cell (hPSC) differentiation efficiency. However, the optimal environmental factors to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Here, we hypothesized that low oxygen exposure enhances the molecular and functional maturity of the cardiomyocytes. We aimed at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hPSCs in 2D- and 3D-based protocols. hPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed. The mitochondrial localization and metabolic properties were evaluated. The intracellular Ca2+ handling and contractile properties were also monitored. The 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac spheroids containing hPSC-CMs in LOE further exhibited cardiac markers, hypertrophy, steadier SR Ca2+ release properties revealing a better SR Ca2+ handling, and enhanced contractile force. Preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac spheroids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.