Highly sensitive detection of low abundant molecules by pyro-electrohydro-dynamic jetting

The effective detection of low-concentrated molecules in small volumes represents a significant challenge in many sectors such as biomedicine, safety, and pollution. Here, we show an easy way to dispense liquid droplets from few μl volume (0.2-0.5 μl) of a mother drop, used as reservoir, by using a pyro-electrohydro-dynamic jetting (p-jet) dispenser. This system is proposed for multi-purpose applications such as printing viscous fluids and as a biosensor system. The p-jet system is based on the pyroelectric effect of polar dielectric crystals such as lithium niobate (LN). The electric field generated by the pyroelectric effect acts electro-hydrodynamically on the sample of liquid, allowing the deposition of small volumes. The p-jet approach allows to obtain the dispensing of drops of very small volumes (up to tenths of a picoliter) avoiding the use of syringes and nozzles generally used in standard technologies. The reliability of the technique as a biosensor is demonstrated both in the case of oligonucleotides and in a sample of clinical interest, namely gliadin. The results show the possibility of detecting these biomolecules even when they are low abundant, i.e. down to attomolar. The results show a marked improvement in the detection limit (LOD) when compared with the conventional technique (ELISA). Moreover, it has been presented the possibility of using the p-jet as a useful tool in the detection of biomarkers, present in the blood but currently not detectable with conventional techniques and related to neurodegenerative diseases such as Alzheimer.


INTRODUCTION
The ability to manipulate and dispense liquids on a micro and nanometric scale allows important benefits in many fields such as combinatorial chemistry, printing applications, and biotechnology. On the other side, the development of increasingly performing and innovative sensors is now the prerogative of research of the most advanced laboratories in the world [1][2][3][4]. In particular, the ability to detect biomolecules in very low concentrations has repercussions in various areas such as biomedicine, pollution, forensic activity etc. [3,4]. Hence, there is a constantly growing demand for specific tools for the deposition of ultra-small quantities of materials in predefined places in order to develop miniaturized instruments for the fabrication of highly integrated and automated "lab-on-a-chip" systems based on microfluidics [5,6]. The ability to fabricate complex microfluidic architectures has allowed scientists to create new highly efficient experimental formats for fast processing of small analytical volumes, thus resulting in reduced reagent consumption and extremely low manufacturing costs. Moreover, greater operational flexibility and the possibility of integrating functional components within complex analytical schemes give the additional benefit of greater portability of the instruments. Currently, most microfluidic systems consist of microchannels in which liquids are manipulated by applying pressure or voltage variations to the electrodes thus involving some disadvantages such as complex manufacturing, cross-contamination of the sample and the need to apply high voltages or pressures. In recent years, conventional approaches such as microcontact printing [7], photolithography [8], nanoimprint [9], and direct laser writing [10] have been overcome by inkjet printing, that exhibits numerous advantages in terms of flexibility, high precision and controllability of nanomaterials patterns in nanofabrication and nanodevices [11]. In particular, Electrohydrodinamic jet (E-jet) printing is widely used in important applications, such as biosensors [12], micro-nano optical devices [13], and flexible electronic devices [14].
Here we show a simple system in which the manipulation and dispensing of the liquid is obtained through an 'electrode-free' configuration based on the physical properties of a dielectric crystal such as the Lithium Niobate-LiNbO3 (LN). The technique, defined as "pyroelectrodynamic dispenser jet" (p-jet), allows to deposit both separate drops with lower volumes than traditional systems, and printing fiber of viscous fluids [15][16][17]. This technique is based on the dispensing induced by means of an electric field generated by the pyroelectric effect from LN crystal subjected to a temperature gradient. P-jet allows to manipulate liquids in a no-contact mode and avoids the use of nozzles and external electrodes thus overcoming some severe limitation of ink-jet printing, ie nozzle clogging [18]. Recently, p-jet approach was used for different applications, including, accumulation of very diluted biomolecule [19,20], the formation of spiral fibers [21] and manipulation of soft matter [15][16][17]. We show the main applications of this technology starting from the manipulation and printing of viscous fluids to the develop of a sensor capable of detecting low concentrations of molecules otherwise not detectable with conventional technologies. In this framework, the efficiency of the p-jet based biosensor for the detection of oligonucleotides and gliadin protein was demonstrated. The usefulness of the p-jet based biosensor can be extended to any field in which the detection of low concentration quantities is required, such as the case of biomarker in the blood (currently not detectable with the technologies available on the market) which represents the alarm bell of the presence of some neurodegenerative disease such as Alzheimer's disease (AD). Therefore, the p-jet proves to be a useful, low cost and easy to implement tool that paves the way for the early diagnosis of many silent diseases.

Lithium niobate
The lithium niobate (LN) crystals were purchased from Crystal Technology Inc. and were in the form of 500 µm thick and 3-inch diameter wafers with both sides polished and mono-domain ferroelectric state.

Pyroelectric effect
LN with cut c is a ferroelectric crystal is characterized by pyroelectric properties, therefore, at room temperature, it has a spontaneous polarization Ps conventionally oriented from the so-called face c to face c +. At equilibrium, if it is not thermally stimulated, the polarization charge of the LN crystal is completely screened from external screening charges on the surface of the crystal and no electric field is generated. The thermal stimulation induces a change of Ps as a consequence, a transient electrostatic state appears with screening charges not compensated on the crystal surface. According to the pyroelectric effect, this phenomenon is associated with a high electric field that originates from the surface of the crystal [22][23][24]. This electric field can be used for a wide range of applications ranging from biological to soft matter manipulation [25][26][27][28][29].  A conventional optical path consisting of a collimated LED, an optical microscope objective (10) and a high-speed CMOS camera (Motion Pro Y3-S1, pixel size of 10.85x10.85 μm 2 ). These instruments allow us to have a side view of the experiment during the dispensing events. The p-jet set-up consists of a basic support that hosts the liquid sample, manually deposited, that we will call here "mother drop" (eg 0.2 µL volume), while the dispensing slide, that represents the target support where the deposition occurs, faces parallel and is mounted on a motorized translation system. The pyroelectric crystal is placed above the dispensing slide substrate. The temperature gradient is induced in the crystal by direct contact with a heating instrument that can be a conducting wire heater (heated by Joule effect), or a hot tip of a common soldering iron or without contact using a laser source that emits in the far-infrared (CO 2 laser, λ= 10.3 µm wavelength falls within the absorption band of the LN crystals).The pyro-electrohydrodynamic effect consists of charge displacement induced in the mother drop due to the electric field, pyroelectrically generated (in Figure 1 the field it is schematized by grey lines), that employs attractive and repulsive forces to the liquid molecules [30,31].

P-jet system
Figures 2 (a) and 2 (b) schematically show the side view of the configuration of the mother drop. Neglecting the effect of gravity, the mother drop has a hemispherical shape with a static contact angle θ defined by the mechanical equilibrium of three interfacial tensions: liquid vapour (σlv), solid vapour (σsv) and solid-liquid (σsl) interface tensions, respectively. Following a thermal stimulation, the LN generates an electric field that surrounds the mother drop which deforms due to the effect of the electrical, capillary and viscous forces, as shown schematically in Fig. 2 (b). Charges accumulate on the surface, and the drop assumes a conical profile called "Taylor cone". This conical shape is maintained up to a certain critical limit of the electric potential [32]. Once that limit is exceeded, the profile of the meniscus can no longer be supported by the surface tension, and the fluid will be ejected from the apex of the cone. The electric field produces an electrostatic polarization stress that causes the initial deformation of the drop, therefore, the fluid will be ejected from the cone when the electrostatic stresses overcome the surface tension [33][34][35].

5'Cy5
(λex ˷ 650 nm; λem ˷ 670 nm) fluorescence-labelled oligonucleotide (5'-CGCGATCCCACCCAGGGAGCCACTGAGATGATGGCG-7', 66.7% G-C content, MW 12,131 gmol -1 ) was synthesized at Eurofins mwg/ operon-Ebersberg -D. region. The oligonucleotide was solubilized in Tris-EDTA buffer at initial concentration of 100 µM . A 300-nM stock solution was prepared and serial dilutions in Tris-EDTA were used. The samples were mixed with an advanced buffer system (Micro-Spotting Solution Plus 2X, ArrayIt), in a ratio 1:1, to improve the surface properties of the oligonucleotide samples deposited during printing. The analyte solution (0.2 ml) was deposited onto the base support by a Hamilton Modified Microliter Syringe (7000 Series).

b. Gliadin
1 mg of gliadin (Sigma Aldrich) was solubilized in 1ml of phosphate-buffered saline. 0.1M sodium bicarbonate solution was added to raise the pH of the reaction mixture to 8-8.5. A volume of 100 µlof solution was labelled using the Alexa Fluor 647 Antibody Labelling Kit (Molecular Probes, Invitrogen) following the manufacturer's instructions. The reaction mixture was loaded onto a spin column (available with the kit), containing a gel filtration resin in phosphate-buffered saline (pH 7.2), and after centrifugation at 1,100g for 5min, the labelled protein (˷100 µl) was collected in a tube, while the band of unreacted dye was retained in the void volume. The concentration of labelled gliadin (610 ng ml -1 ) was calculated measuring the adsorbance at 280 nm and subtracting the adsorbance at 650 nm corrected by a 0.03 factor, to eliminate the contribution of lysine -linked dye.

Fluorescence scanner
A fluorescence scanner InnoScan 710 (Innopsys, Carbonne, France) was used for reading the slides. Main scan parameters: laser source at 530 nm and 10mW power; pixel size 3 mm and scan speed of 10 lines s -1 ; detection gain 25% PMT

The p-jet for liquid printing
The p-jet configuration allows printing various liquids, such as viscous fluids. Given the volume (V) of the mother drop, which acts as a mother drop, and its contact angle (θ), depending on the type of liquid and the substrate, a critical distance value DC, between the mother drop and the deposition slide, can be defined: According to this equation, it is possible to distinguish two different conditions: (1) for distances shorter than DC a stable liquid bridge is generated and it is therefore possible to print a strip of fluid continuously by translation of the dispensing slide; (2) for distances larger than DC, an instability situation develops and small daughter droplets will be thrown from the mother drop. Therefore, this instability is used to dose and dispense liquids [34]. The p-jet system can be used as a printing tool directly to the desired substrate by break the mother drop in secondary droplets to be delivered as separate single drops or having a continuous deposition of the fluid strip starting from the mother drop.
We are able to print different geometries by controlling the direction of translation and the speed of the deposition slide and droplets and lines of liquids with extremely regular diameters and widths and according to a wide variety of geometries. Figure 3 shows some simple patterns printed by oleic acid, almond oil and mineral oil in separate droplets, straight and curved lines. The revolutionary feature of p-jet is to avoid the use of the deposition tools traditionally used in printing protocols (i.e. syringes, nozzles), thus performing much easier and faster dosing procedures.

The p-jet for accumulating biomolecules
The reliability and the multi-purpose capability of the p-jet technique was demonstrated for biosensing application. The p-jet system is able to concentrate low abundant biomolecules by guiding and accumulating biomolecules in the mother drop directly onto the surface of a functionalized deposition slide. The key concept of this type of approach is called "split and stack". The biomolecules are dispensed on a small area of the deposition slide and this occurs thanks to the possibility of split the mother drop through a series of very small droplets and stuck them in a limited area of the receiving substrate. Using labelled biomolecules, with this approach, it is possible to increase the density of the fluorescence signal and improve significantly the sensitivity. Furthermore, the classic diffusion phenomena, encountered in conventional detection techniques such as ELISA, are avoided [36]. The comparison between the two approach is schematized in fig 4. The colored dots represent the target molecules and other antigenes in a solution, while the Y symbols indicate the binding molecules on the deposition slide (SuperAmmine, Super Nylon, ArrayIt). We focus here on a sample solution with critical concentration, namely well below the values corresponding to the LOD of standard ELISA kits (tens of pg/mL). Moreover, for the sake of simplicity, we hypothesize here that the reaction between deposition slide and binding molecules produces a fluorescent (FL) spot. The well-based reaction (ELISA kit) is shown in Fig.4(a) while the p-jet based reaction is shown in Fig.4(b). In the first case, following a certain incubation time, the molecules to be detected, diffuse in the reaction volume (100 µL) until they meet the binding molecules on the surface of the well. However, there is a relatively high discrepancy between the volumetric reaction and the concentration of molecules, the successful bonds are distributed over a relatively large surface area, thus producing a FL signal per unit area, very weak with a consequent signal-to-noise ratio (SNR) unsatisfactory which makes the detection of molecules inadequate. On the contrary, the p-jet allows us to dispense the same quantity of molecules on a very confined area of the target slide (~ 1 ÷ 5 µm 2 ) thus forcing the target molecules to react with the binding ones in a reduced volume. Consequently, the FL signal per area unit is much higher, the SNR increases significantly and even in the case of low abundant molecules it is possible to detect them.

Oligonucleotides detection
The ability of the system to concentrate the analytes has been deepened by comparing the fluorescence signal of the pjet spots and that of the spots obtained by depositing the same sample volume with a standard pipette. As first test, we used a stock solution of a single strand (SS, single strand) DNA oligonucleotide (15-20 bases) at 300 nM concentration labeled with Cy5 (fluorophore emitting in the red region λex ~ 650 nm; λem ~ 670 nm). Successive dilutions from that solution were prepared. Serial dilutions, between 300 nM and 300 aM have been analyzed, and Figure 5 (a) shows how by depositing the drop in the classical way, the fluorescence signal decays with decreasing concentration. The limit of detection (LOD) was calculated using a value three times the intensity of the standard deviation of the background. Figure 5

Gliadin detection
The performance of the p-jet for detecting low abundant molecules was tested also in case of a protein of clinical interest, the gliadin. Gliadin represents the predominant protein component in gluten. The presence of gluten in food must be kept under control by people with celiac disease who follow a gluten-free diet. In the European Union, a maximum level of 20 p.p.m. of gluten is allowed for a product declared "gluten-free". To date, the presence of gluten in food is usually detected by conventional ELISA tests [37]. As already said, this technology shows diffusion limits that do not allow the detection of biomolecules when they are in very low concentration. Therefore, a reliable and highly sensitive technique for evaluating the gluten content in foods it would be of great benefit to patients, dieters and food producers.
The p-jet was used to detect different concentrations of gliadin labelled by Alexa Fluor 647 (240pg / ml; 120pg / ml; 24pg / ml; 12 pg / ml; 1 pg / ml). Figure 6a shows the fluorescence signal of the spots obtained with p-jet technology as a function of the dilution factor of gliadin, while Fig. 6b shows the corresponding fluorescence profiles and scanner images of the spots obtained with the p-jet system. The results clearly show that the p-jet system can efficiently detect 1 pg of gliadin in 0.2 µl of solution (0.005 p.p.m.), thus achieving a 60-fold improvement over the current ELISA test limits (0.3 p.p.m.) [38].

AD biomarker
In the framework of biosensors, the detection of biomarkers present in very low concentrations in the blood could have significant implications in the early diagnosis of some diseases, especially in the neurological field as in case of the Alzheimer's disease [39]. The current guidelines for clinical diagnosis of AD establish the determination of specific protein biomarkers (Amyloid-beta, tau, P-tau) in cerebrospinal fluid (CSF) through ELISA kit and the positron emission tomography (PET) of the brain with amyloid tracer. This procedure, besides being invasive for the patient, requires hospitalization and qualified personnel, and, moreover the disease is diagnosed when has progressed massively. Nowadays the traditional ELISA kits cannot determine such biomarkers in peripheral blood due to their abundance well below his LOD of 50-100 pg/mL. This detection limit (LOD) is due to the diffusion phenomena encountered by antigens and antibodies due to the large reaction volumes (50-100uL) in relatively large areas (1/5 mm 2 ), leading to a significant lowering of the signal / noise ratio.
The use of p-jet as biosensor allows us to detect small traces in the blood (concentration below 1pg / mL) of protein biomarkers of AD. In order to overcome the ELISA LOD, the biosensor aims to concentrate micro-droplets of the biological sample, containing the biomarkers of interest, on a very restricted area of a deposition slide chemically bonding the biomarker with its antibody. Thus the diffusion problems are avoided because the reaction between the biomarker (antigen) and the respective antibody is maximized, thus maximizing the probability of encounter, thus increasing the fluorescence signal per unit area. The volumes to be used are clearly lower than those used in standard technology, in fact 0.2 µL of biological sample containing the biomolecules of interest (marked in fluorescence) are deposited manually by an operator on a support that acts as a mother drop. The deposition slide is chemically functionalized with the antibody (antibody specific to the biomolecule to be detected) and placed as showed schematically in Fig.4 (b). After thermal stimulation, it follows on that a pyro-electric field appears capable of dividing the mother drop into many small micro-drops which will be accumulated in a single site of micrometric dimension on the functionalized deposition slide. Therefore, the reaction will be forced with a high efficiency since the diffusion phenomena will be cancelled obtaining much stronger fluorescence signals. The authors are leading the European Project 'SensApp' funded under the H2020 program and aiming at developing a super-sensor able to detect AD biomarkers by a simple blood test [40]. Thanks to this approach it will possible to develop a simple and inexpensive test to identify a biomarker panel directly in a drop of blood where the concentration of biomolecules indicative of the disease is very low. Therefore, it will be possible to carry out an early diagnosis of AD and easy screening among the population.

CONCLUSION
The innovative pyro-electrodynamic jet (p-jet) method has been used as a printing device for viscous fluids and for applications as a biosensor. The p-jet is based on the hydro-electrodynamic effect on the fluid, generated by an electric field activated pyroelectrically by a lithium niobate crystal. The performances of the p-jet have been tested for the rapid and extremely sensitive detection of oligonucleotides and gliadin protein. The operating principle is extremely simple and effective. A mother drop containing the biomolecules to be detected is split into many small droplets and stacked in a limited area of the deposition slide, in this way, the diffusion phenomena that limit the detection of low abundant molecules are eliminated and therefore sensitivity is increased. Furthermore, this innovative system is extremely versatile since it can also be used (following appropriate adaptations) to identify biomarkers present in very low concentrations in the blood related to pathologies such as Alzheimer's disease. It is easy to predict the social impact of this advanced technology, since, as in the case of AD disease, which has a high incidence and is currently without a cure, its early diagnosis would lead to an advanced study of the disease in the early stages of human beings and therefore the identification of possible treatments. Furthermore, the technique is extremely simple and inexpensive and absolutely non-invasive for the patient. Hospitalization and withdrawal of the patient's spinal fluid with very delicate procedures that require specialized and qualified personnel is not necessary. This technology will enable faster and non-invasive early diagnosis of diseases in the future simply through routine blood tests, thus paving the way for highly efficient screening programs among the population. In summary, this multipurpose technology opens the way to all those applications where the manipulation of small volumes of fluids is necessary and the need to detect low abundant molecules with important repercussions in the diagnostic field.