ROSANNA TOSCANO, BUSINESS DEVELOPER MEDTECH, FEMTOPRINT, ANDREA LOVERA, CHIEF SCIENCE OFFICER AND HEAD OF SOLUTION DIVISION, FEMTOPRINT, ROBERTA CALMO, DEPARTMENT OF APPLIED SCIENCE AND TECHNOLOGY (DISAT), POLITECNICO DI TORINO, AND STEFANO STASSI, DISAT, POLITECNICO DI TORINO
Over the last decade, biological and chemical research has moved in the direction of fast and highly sensitive molecular analysis. Among the traditional biosensing platforms, mechanical resonators have shown potential for rapid cell analysis, enabling detection of small masses as a function of changes in their mechanical properties. These resonators operate in a dynamic configuration oscillating at a characteristic resonance frequency that is proportional to the resonator mass. The addition of new mass to the resonator, for example, from cells or bacteria, causes a shift in frequency directly proportional to the added mass. The use of resonators in the biosensing field has increased, thanks in part to their remarkable ability to detect label-free mass variations associated with biomolecular recognition events, which typically range from nanomolar to femtomolar concentrations.
FEMTOprint—a contract manufacturer of 3D printed microdevices in glass—and the Department of Applied Science and Technology (DISAT) at the Politecnico di Torino (Turin Polytechnic) recently developed a class of suspended microchannel resonators (SMRs) that is able to discriminate between healthy and tumour cells through resonance shift analysis of an internal oscillator. These SMRs consist of a microfluidic channel embedded in a vibrating cantilever under vacuum, as shown in figure 1. They afford high-resolution measurement of density, viscosity and specific gravity of fluids, as well as simultaneous detection of both position and velocity of target analytes through the monitoring of multiple resonance frequencies.
1 of 2
Figure 1a
A suspended microchannel resonator (SMR) produced by FEMTOprint and the Politecnico di Torino (Turin Polytechnic). This class of SMRs allows for early detection of tumour cells in the blood stream through the analysis of differences in cell deformability. (a) Schematic representation of the SMR design showing the buried channel and embedded inlets. (b) SEM images of an as-fabricated glass SMR, with the suspended bridge and the embedded channel inlets highlighted in yellow. (c) An SMR prototype coupled to microfluidic tubing for testing and evaluation.*Figure 1b: Reprinted from Sensors and Actuators B: Chemical, volume 283, Calmo, R., Lovera, A., Stassi, S., Chiadò, A., Scaiola, D., Bosco, F. and Ricciardi, C., Monolithic glass suspended microchannel resonators for enhanced mass sensing of liquids, pp.298–303, Copyright 2019, with special permission from Elsevier.
2 of 2
Figure 1b
A suspended microchannel resonator (SMR) produced by FEMTOprint and the Politecnico di Torino (Turin Polytechnic). This class of SMRs allows for early detection of tumour cells in the blood stream through the analysis of differences in cell deformability. (a) Schematic representation of the SMR design showing the buried channel and embedded inlets. (b) SEM images of an as-fabricated glass SMR, with the suspended bridge and the embedded channel inlets highlighted in yellow. (c) An SMR prototype coupled to microfluidic tubing for testing and evaluation.*Figure 1b: Reprinted from Sensors and Actuators B: Chemical, volume 283, Calmo, R., Lovera, A., Stassi, S., Chiadò, A., Scaiola, D., Bosco, F. and Ricciardi, C., Monolithic glass suspended microchannel resonators for enhanced mass sensing of liquids, pp.298–303, Copyright 2019, with special permission from Elsevier.
Figure 1c
A suspended microchannel resonator (SMR) produced by FEMTOprint and the Politecnico di Torino (Turin Polytechnic). This class of SMRs allows for early detection of tumour cells in the blood stream through the analysis of differences in cell deformability. (a) Schematic representation of the SMR design showing the buried channel and embedded inlets. (b) SEM images of an as-fabricated glass SMR, with the suspended bridge and the embedded channel inlets highlighted in yellow. (c) An SMR prototype coupled to microfluidic tubing for testing and evaluation.*Figure 1b: Reprinted from Sensors and Actuators B: Chemical, volume 283, Calmo, R., Lovera, A., Stassi, S., Chiadò, A., Scaiola, D., Bosco, F. and Ricciardi, C., Monolithic glass suspended microchannel resonators for enhanced mass sensing of liquids, pp.298–303, Copyright 2019, with special permission from Elsevier.
The ability of the SMRs to monitor small variations directly in a liquid environment means that they are an advantageous alternative to more traditional biosensing technologies. Indeed, they are an extremely attractive solution for integration into lab-on-a-chip devices for clinical analysis and new flow-through measurements, providing, for example, early detection of tumour cells in the blood stream through discrimination of differences in cell deformability. This is possible by introducing a small bottleneck inside the SMR and monitoring the transition time of healthy and malignant cells1.
Another promising application is the prediction of therapeutic efficacy using ex-vivo cells through investigation of different therapeutic treatments or dosage. SMRs can be used to select the most effective treatment by measuring the mass accumulation rate (MAR), a technique often applied to tumour cells2.
Fused silica microdevices enabled by femtosecond laser micromachining
The widespread use of SMRs has been limited by traditional fabrication processes, which involve photolithography and etching, using materials such as silicon, silicon oxide and silicon nitride. Although this fabrication process is effective, it is often challenging, time-consuming and expensive, requiring long development cycles to ensure optimisation. Additionally, the multi-step nature of the process requires a cleanroom environment to ensure successful fabrication of functional, defect-free devices.
Recent improvements in femtosecond laser micromachining have enabled fabrication of micro-scale devices in glass, combining structural features with optical and micromechanical functionalities. FEMTOprint and the Politecnico di Torino used the FEMTOPRINT femtosecond laser 3D printing process shown in figure 2 to fabricate the aforementioned SMRs in glass.
Figure 2
The FEMTOPRINT femtosecond laser exposure process. (a) Schematic representation of the SMR fabrication process, which includes the 3D laser patterning of the suspended resonator with embedded microfluidic channel, and release of the structure by means of a mask-free etching step. (b) Table listing the principal characteristics of the FEMTOPRINT technology.
Femtosecond laser 3D printing technology for medical microsystems
FEMTOPRINT is a subtractive, direct-write microfabrication process, exploiting femtosecond laser 3D printing and etching solutions to create advanced 3D microsystems with micrometre precision and repeatability. The process works by taking advantage of highly localised material modifications, triggered by non-linear absorption during the laser exposure step of fabrication. These localised modifications are easily dissolved in etching solutions, with selectivity of up to 1,000:1 compared with bulk un-modified glass.
FEMTOPRINT can be used in many medical fields to combine fluidic, mechanical, optical and electrical functionalities in application-specific devices such as:
- adhesive-free, biocompatible glass-to-glass implants;
- drug delivery systems;
- lab- and organ-on-chips;
- microelectrode arrays (MEAs);
- microlens arrays (MLAs) for optical sensing and detection;
- micropore membranes and mixing systems;
- scaffolds for 3D cell cultures;
- sensor technology systems and microelectromechanical systems (MEMS); and
- mould masters for replication.
Some of these examples are shown in figure 3.
Figure 3
FEMTOPRINT technology applications. Examples of advanced 3D microsystems printed using the FEMTOPRINT process. (a) Safe puncture optimised tool for retinal vein cannulation (SPOT-RVC) for injecting a clot-dissolving drug directly into the retinal veins to treat retinal vein occlusion. EPFL (Swiss Federal Institute of Technology Lausanne), Hôpital ophtalmique Jules-Gonin and Innosuisse, Switzerland. (b) Opto-fluidic lab-on-a-chip with integrated lenses for chemical analysis of particles. CEA (French Alternative Energies and Atomic Energy Commission), France. (c) Lab-on-a-chip with integrated gold electrodes, featuring a nanolitre droplet generator for single cell encapsulation. Shilps Sciences, India. (d) Scaffolds with micron-scale features for faster, higher-quality 3D cultures of cells and organoids. INRA (National Institute of Agronomic Research), France.
Design and prototyping of a glass SMR
To test the FEMTOPRINT technology for the fabrication of the glass SMR, a clamped beam design was chosen. Different dimensions were tested for beam length (250– 1,000 μm), width (50–75 μm) and embedded channel dimension (10, 30 and 55 μm). The channel shape was strongly dependent on the laser interaction volume, as shown in figure 4.
Figure 4
Scanning electron microscope (SEM) images showing various cross-section views of the embedded channel during prototyping. The channel shape is strongly dependent on the size and shape of the laser interaction volume.
The FEMTOPRINT technology enabled fast production of a monolithic SMR without residual stress, which is a common issue in multi-step processes that compromises the mechanical performance of the sensor. The resonance properties of the fabricated SMR were accurately characterised in terms of Allan deviation, frequency and quality factor, which are useful parameters for evaluation of the mass and density responsivity, as well as the minimum detectable mass, as shown in figure 5.
Figure 5
Mechanical performance of the glass SMR. (a) Responsivity versus SMR mass plot, showing the SMR behaviour as a function of the device dimensions and mass. (b) Table listing the principal figures of merit, which describe the sensing properties of the glass SMR.
Innovations in glass resonator technology can meaningfully affect the field of biomedical diagnostics
Testing revealed that the monolithic glass SMRs could differentiate liquids of varying mass density down to a resolution of 1.04 x 10−3 kg/m3, a result comparable to that of silicon-based SMRs, and outperform commercial microcapillary glass resonators. Additionally, the FEMTOPRINT technology proved a faster and more cost-effective fabrication method, as well as introduced novel 3D features.
The effective biosensing capability of the SMR was demonstrated by evaluating the microbial load in aqueous solutions containing different concentrations of the bacterium pseudomonas fluorescens, selected on account of its being the closest relative to the pathogenic pseudomonas aeruginosa, frequently responsible for multi-drug resistance in nosocomial infections. The glass SMR could distinguish different sample dilutions with high confidence and repeatability, resulting in a detection limit of around 60 picograms, or approximately 150 bacteria, as shown in figure 6.
Figure 6
Bacteria quantity in an aqueous solution, monitored by evaluating the mass load variation inside the SMR as a function of the frequency shift.
The testing confirmed that glass SMRs can be exploited for real-time monitoring of bacteria with enhanced performance, improving healthcare and food safety. SMR device transparency provides an opportunity for monitoring the channel using imaging techniques, thus paving the way for a new methodology of integrated microgravimetric and optical analyses of cell behaviour.
Improvement of mass resolution for new crucial biological applications that can be introduced in the points of care
The FEMTOPRINT technology can bring micro-based devices from a few samples in a research lab to industrial serial production, improving point of care diagnostics services. A future target of this project is the enhancement of the mass resolution of the SMR to approach new, crucial biological applications such as monitoring cell growth, survival, division and response to external stimuli. Sensitivity enhancement will allow: the detailed investigation of specific cell response to drug treatments; the preliminary screening for diseases affecting the plasticity of cells such as cancer; and the investigation of biomolecular cell composition, for example, the study of DNA, RNA and proteins.
FEMTOprint
DISAT, Politecnico di Torino
References:
1Shaw Bagnall, J., Byun, S., Miyamoto, D. T., Kang, J. H., Maheswaren, S., Stott, S. L., Toner, M. and Manalis, S. R. (2016). Deformability-based cell selection with downstream immunofluorescence analysis. Integrative Biology, volume 8, issue 5, pp.654–664.
2Cetin, A. E., Stevens, M. M., Calistri, N. L., Fulciniti, M., Olcum, S., Kimmerling, R. J., Munshi, N. C. and Manalis, S. R. (2017). Determining therapeutic susceptibility in multiple myeloma by single-cell mass accumulation. Nature Communications, volume 8, article no. 1613.